32142, 21481, 25964, 21686, novel dehydrogenase molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated DHDR nucleic acid molecules, which encode novel DHDR-related dehydrogenase molecules. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing DHDR nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a DHDR gene has been introduced or disrupted. The invention still further provides isolated DHDR proteins, fusion proteins, antigenic peptides and anti-DHDR antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/664,506, filed Sep. 17, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/838,561, filed Apr. 18, 2001, now granted asU.S. Pat. No. 6,627,423; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/816,760, filed Mar. 23, 2001, now granted asU.S. Pat. No. 6,613,555; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/634,955, filed Aug. 8, 2000, now granted as U.S.Pat. No. 6,511,834; which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/192,002, filed Mar. 24, 2000, now abandoned. Theentire contents of all of the above-referenced applications areincorporated herein by this reference.

The contents of the Sequence Listing are submitted herewith on compactdisc in duplicate. Each disc has a copy of the file “sequencelisting.txt,” which is incorporated herein by this reference. This fileis 104 kilobytes and is a copy of the Supplemental Sequence Listingfiled on Aug. 2, 2005 in U.S. patent application Ser. No. 10/664,506,filed Sep. 17, 2003. This file was copied onto compact disc on Jan. 17,2006.

BACKGROUND OF THE INVENTION

The oxidation and reduction of molecules is of critical importance inmost metabolic and catabolic pathways in cells. A large family ofenzymes which facilitate these molecular alterations, termeddehydrogenases, have been identified. In the forward reaction, theseenzymes catalyze the transfer of a hydride ion from the target substrateto the enzyme or a cofactor of the enzyme (e.g., NAD⁺ or NADP⁺), therebyforming a carbonyl group on the substrate. These enzymes are also ableto participate in the reverse reaction, wherein a carbonyl group on thetarget molecule is reduced by the transfer of a hydride group from theenzyme. Members of the dehydrogenase family are found in nearly allorganisms, from microbes to Drosophila to humans. Both between speciesand within the same species, dehydrogenases vary widely, and structuralsimilarities between distant dehydrogenase family members are mostfrequently found in the cofactor binding site of the enzyme. Even withina particular subclass of dehydrogenase molecules, e.g., the short-chaindehydrogenase molecules, members typically display only 15-30% aminoacid sequence identity, and this is limited to the cofactor binding siteand the catalytic site (Jornvall et al. (1995) Biochemistry34:6003-6013).

Different classes of dehydrogenases are specific for an array ofbiological and chemical substrates. For example, there existdehydrogenases specific for alcohols, for aldehydes, for steroids, andfor lipids, with particularly important classes of dehydrogenasesincluding the short-chain dehydrogenase/reductases, the medium-chaindehydrogenases, the aldehyde dehydrogenases, the alcohol dehydrogenases,and the steroid dehydrogenases. Within each of these classes, eachenzyme is specific for a particular substrate (e.g., ethanol orisopropanol, but not both with equivalent affinity). This exquisitespecificity not only permits tight regulation of the metabolic andcatabolic pathways in which these enzymes participate, without affectingsimilar but separate biochemical pathways in the same cell or tissue.The short-chain dehydrogenases, part of the alcohol oxidoreductasesuperfamily (Reid et al. (1994) Crit. Rev. Microbiol. 20:13-56), areZn⁺⁺-independent enzymes with an N-terminal cofactor binding site and aC-terminal catalytic domain (Persson et al. (1995) Adv. Exp. Med. Biol.372:383-395; Jornvall et al. (1995) supra), whereas the medium chaindehydrogenases are Zn⁺⁺-dependent enzymes with an N-terminal catalyticdomain and a C-terminal coenzyme binding domain (Jornvall et al. (1995)supra; Jornvall et al. (1999) FEBS Lett. 445:261-264). The steroiddehydrogenases are a subclass of the short-chain dehydrogenases, and areknown to be involved in a variety of biochemical pathways, affectingmammalian reproduction, hypertension, neoplasia, and digestion (Duax etal. (2000) Vitamins and Hormones 58:121-148). Aldehyde dehydrogenasesshow heterogeneity in the placement of these domains, and alsoheterogeneity in their substrates, which include toxic substances,retinoic acid, betaine, biogenic amine, and neurotransmitters (Hsu etal. (1997) Gene 189:89-94). It is common in higher organisms fordifferent dehydrogenase molecules to be expressed in different tissues,according to the localization of the substrate for which the enzyme isspecific. For example, different mammalian aldehyde dehydrogenases arelocalized to different tissues, e.g., salivary gland, stomach, andkidney (Hsu et al. (1997) supra).

Dehydrogenases play important roles in the production and breakdown ofnearly all major metabolic intermediates, including amino acids,vitamins, energy molecules (e.g., glucose, sucrose, and their breakdownproducts), signal molecules (e.g., transcription factors andneurotransmitters), and nucleic acids. As such, their activitycontributes to the ability of the cell to grow and differentiate, toproliferate, and to communicate and interact with other cells.Dehydrogenases also are important in the detoxification of compounds towhich the organism is exposed, such as alcohols, toxins, carcinogens,and mutagens.

A dehydrogenase of the short-chain family, 11-beta-hydroxysteroiddehydrogenase, activates glucocorticoids in the liver. Glucocorticoidsare known to induce transcription of hepatitis B virus (HBV) genes,probably by direct binding of the ligand-glucocorticoid receptor complexto an enhancer element in the HBV genome. There is also evidence thatshort chain dehydrogenases are transcriptional cofactors for retrovirusgene activation.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel members of the family of dehydrogenase molecules, referred toherein as DHDR nucleic acid and protein molecules (e.g., DHDR-1, DHDR-2,DHDR-3, and DHDR-4). The DHDR nucleic acid and protein molecules of thepresent invention are useful as modulating agents in regulating avariety of cellular processes, e.g., viral infection, cellularproliferation, growth, differentiation, and/or migration. Accordingly,in one aspect, this invention provides isolated nucleic acid moleculesencoding DHDR proteins or biologically active portions thereof, as wellas nucleic acid fragments suitable as primers or hybridization probesfor the detection of DHDR-encoding nucleic acids.

In one embodiment, a DHDR nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 89%, 90%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, 99.99% or more identical to the nucleotide sequence (e.g., to theentire length of the nucleotide sequence) shown in SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845, or acomplement thereof.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14,or 16, or a complement thereof. In another embodiment, the nucleic acidmolecule includes SEQ ID NO:3 and nucleotides 1-62 of SEQ ID NO:1. Inanother embodiment, the nucleic acid molecule includes SEQ ID NO:6 andnucleotides 1-330 of SEQ ID NO:4. In yet another embodiment, the nucleicacid molecule includes SEQ ID NO:9 and nucleotides 1-280 of SEQ ID NO:7.In another embodiment, the nucleic acid molecule includes SEQ ID NO:12and nucleotides 1-60 of SEQ ID NO:10. In another embodiment, the nucleicacid molecule includes SEQ ID NO:16 and nucleotides 1-101 of SEQ IDNO:14. In yet a further embodiment, the nucleic acid molecule includesSEQ ID NO:3 and nucleotides 2469-2660 of SEQ ID NO:1. In anotherembodiment, the nucleic acid molecule includes SEQ ID NO:6 andnucleotides 1264-1379 of SEQ ID NO:4. In another embodiment, the nucleicacid molecule includes SEQ ID NO:9 and nucleotides 1388-1725 of SEQ IDNO:7. In another embodiment, the nucleic acid molecule includes SEQ IDNO:12 and nucleotides 1027-1209 of SEQ ID NO:10. In still anotherembodiment, the nucleic acid molecule includes SEQ ID NO:16 andnucleotides 1035-1108 of SEQ ID NO:14. In another preferred embodiment,the nucleic acid molecule consists of the nucleotide sequence shown inSEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16.

In another embodiment, a DHDR nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently identical to the amino acid sequence of SEQ ID NO:2, 5, 8,11, or 15, or an amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845. In a preferredembodiment, a DHDR nucleic acid molecule includes a nucleotide sequenceencoding a protein having an amino acid sequence at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to the entire length of the amino acid sequence of SEQ IDNO:2, 5, 8, 11, or 15, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number PTA-1845.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human DHDR-1, DHDR-2, DHDR-3, orDHDR-4. In another preferred embodiment, an isolated nucleic acidmolecule encodes the amino acid sequence of mouse DHDR-2. In yet anotherpreferred embodiment, the nucleic acid molecule includes a nucleotidesequence encoding a protein having the amino acid sequence of SEQ IDNO:2, 5, 8, 11, or 15, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number PTA-1845.In yet another preferred embodiment, the nucleic acid molecule is atleast 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000 or more nucleotides in length. In a further preferredembodiment, the nucleic acid molecule is at least 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or morenucleotides in length and encodes a protein having a DHDR activity (asdescribed herein).

Another embodiment of the invention features nucleic acid molecules,preferably DHDR nucleic acid molecules, which specifically detect DHDRnucleic acid molecules relative to nucleic acid molecules encodingnon-DHDR proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000 or more nucleotides in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence shown in SEQ ID NO:1, 4, 7, 10, or14, the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number PTA-1845, or a complement thereof.

In preferred embodiments, the nucleic acid molecules are at least 15(e.g., 15 contiguous) nucleotides in length and hybridize understringent conditions to the nucleotide molecules set forth in SEQ IDNO:1, 4, 7, 10, or 14.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, or an amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number PTA-1845, wherein the nucleic acid molecule hybridizesto a complement of a nucleic acid molecule comprising SEQ ID NO:1, 3, 4,6, 7, 9, 10, 12, 14, or 16 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a DHDR nucleic acid molecule, e.g., thecoding strand of a DHDR nucleic acid molecule.

Another aspect of the invention provides a vector comprising a DHDRnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably a DHDR protein, by culturingin a suitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the protein is produced.

Another aspect of this invention features isolated or recombinant DHDRproteins and polypeptides. In one embodiment, an isolated DHDR proteinincludes at least one or more of the following domains: a transmembranedomain, a signal peptide domain, an aldehyde dehydrogenaseoxidoreductase domain, an aldehyde dehydrogenase family domain, a shortchain dehydrogenase domain, an oxidoreductase protein dehydrogenasedomain, a 3-beta hydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain.

In a preferred embodiment, a DHDR protein includes at least one or moreof the following domains: a transmembrane domain, a signal peptidedomain, an aldehyde dehydrogenase oxidoreductase domain, an aldehydedehydrogenase family domain, a short chain dehydrogenase domain, anoxidoreductase protein dehydrogenase domain, a 3-beta hydroxysteroiddehydrogenase domain, a NAD-dependent epimerase/dehydratase domain, ashort chain dehydrogenase/reductase domain, a shikimate 5-dehydrogenasedomain, a dehydrogenase domain, and/or a glucose-1-dehydrogenase domain,and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%,68%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, orthe amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number PTA-1845.

In another preferred embodiment, a DHDR protein includes at least one ormore of the following domains: a transmembrane domain, a signal peptidedomain, an aldehyde dehydrogenase oxidoreductase domain, an aldehydedehydrogenase family domain, a short chain dehydrogenase domain, anoxidoreductase protein dehydrogenase domain, a 3-beta hydroxysteroiddehydrogenase domain, a NAD-dependent epimerase/dehydratase domain, ashort chain dehydrogenase/reductase domain, a shikimate 5-dehydrogenasedomain, a dehydrogenase domain, and/or a glucose-1-dehydrogenase domain,and has a DHDR activity (as described herein).

In yet another preferred embodiment, a DHDR protein includes at leastone or more of the following domains: a transmembrane domain, a signalpeptide domain, an aldehyde dehydrogenase oxidoreductase domain, analdehyde dehydrogenase family domain, a short chain dehydrogenasedomain, an oxidoreductase protein dehydrogenase domain, a 3-betahydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain, and is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, whereinthe fragment comprises at least 16 amino acids (e.g., contiguous aminoacids) of the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, or anamino acid sequence encoded by the DNA insert of the plasmid depositedwith the ATCC as Accession Number PTA-1845. In another embodiment, aDHDR protein has the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or15.

In another embodiment, the invention features a DHDR protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, 99.99% or more identical to a nucleotide sequence of SEQID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or a complement thereof. Thisinvention further features a DHDR protein which is encoded by a nucleicacid molecule consisting of a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or a complement thereof.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-DHDR polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind proteins ofthe invention, preferably DHDR proteins. In addition, the DHDR proteinsor biologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a DHDR nucleic acid molecule, protein, or polypeptide ina biological sample by contacting the biological sample with an agentcapable of detecting a DHDR nucleic acid molecule, protein, orpolypeptide such that the presence of a DHDR nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of DHDR activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofDHDR activity such that the presence of DHDR activity is detected in thebiological sample.

In another aspect, the invention provides a method for modulating DHDRactivity comprising contacting a cell capable of expressing DHDR with anagent that modulates DHDR activity such that DHDR activity in the cellis modulated. In one embodiment, the agent inhibits DHDR activity. Inanother embodiment, the agent stimulates DHDR activity. In oneembodiment, the agent is an antibody that specifically binds to a DHDRprotein. In another embodiment, the agent modulates expression of DHDRby modulating transcription of a DHDR gene or translation of a DHDRmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand of aDHDR mRNA or a DHDR gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant or unwantedDHDR protein or nucleic acid expression or activity by administering anagent which is a DHDR modulator to the subject. In one embodiment, theDHDR modulator is a DHDR protein. In another embodiment the DHDRmodulator is a DHDR nucleic acid molecule. In yet another embodiment,the DHDR modulator is a peptide, peptidomimetic, or other smallmolecule. In a preferred embodiment, the disorder characterized byaberrant or unwanted DHDR protein or nucleic acid expression is adehydrogenase-associated disorder, e.g., a viral disorder, a CNSdisorder, a cardiovascular disorder, a muscular disorder, or a cellproliferation, growth, differentiation, or migration disorder.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aDHDR protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a DHDR protein, wherein a wild-typeform of the gene encodes a protein with a DHDR activity.

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of a DHDR protein, byproviding an indicator composition comprising a DHDR protein having DHDRactivity, contacting the indicator composition with a test compound, anddetermining the effect of the test compound on DHDR activity in theindicator composition to identify a compound that modulates the activityof a DHDR protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the cDNA sequence and predicted amino acid sequenceof human DHDR-1 (clone FBH32142). The nucleotide sequence corresponds tonucleic acids 1 to 2660 of SEQ ID NO:1. The amino acid sequencecorresponds to amino acids 1 to 802 of SEQ ID NO:2. The coding regionwithout the 3′ untranslated region of the human DHDR-1 gene is shown inSEQ ID NO:3.

FIG. 2 depicts a structural, hydrophobicity, and antigenicity analysisof the human DHDR-1 protein.

FIG. 3 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of one“transmembrane domains” in the human DHDR-1 protein (SEQ ID NO:2).

FIG. 4 depicts the results of a search which was performed against theHMM database and which resulted in the identification of an “aldehydedehydrogenase family domain” in the human DHDR-1 protein. The aldehydedehydrogenase family domain (SEQ ID NO:17) is aligned with DHDR-1protein (32142, SEQ ID NO:2) residues.

FIGS. 5A-5B depict the results of a search which was performed againstthe ProDom database and which resulted in the identification of a“aldehyde dehydrogenase oxidoreductase domain” in the human DHDR-1protein (SEQ ID NO:2). The aldehyde dehydrogenase oxidoreductase domain(SEQ ID NO:18) is aligned with DHDR-1 protein (Sbjct, SEQ ID NO:2)residues.

FIGS. 6A-6B depict the cDNA sequence and predicted amino acid sequenceof human DHDR-2 (clone Fbh21481). The nucleotide sequence corresponds tonucleic acids 1379 of SEQ ID NO:4. The amino acid sequence correspondsto amino acids 1 to 311 of SEQ ID NO:5. The coding region without the 3′untranslated region of the human DHDR-2 gene is shown in SEQ ID NO:6.

FIG. 7 depicts a structural, hydrophobicity, and antigenicity analysisof the human DHDR-2 protein.

FIG. 8 depicts the results of a signal peptide prediction and a searchwhich was performed against the MEMSAT database and which resulted inthe identification of a signal peptide and one “transmembrane domain” inthe human DHDR-2 protein (SEQ ID NO:5).

FIG. 9 depicts the results of a search which was performed against theHMM database and which resulted in the identification of a “short-chaindehydrogenase domain” in the human DHDR-2 protein. The short chaindehydrogenase domain (SEQ ID NO:19) is aligned with DHDR-2 protein(21481; SEQ ID NO:5) residues.

FIG. 10 depicts the results of a search which was performed against theProDom database and which resulted in the identification of a“oxidoreductase protein dehydrogenase domain” in the human DHDR-2protein (SEQ ID NO:5). The oxidoreductase protein dehydrogenase domain(SEQ ID NO:21) is aligned with DHDR-2 protein residues.

FIGS. 11A-11B depict the cDNA sequence and predicted amino acid sequenceof human DHDR-3 (clone Fbh25964). The nucleotide sequence corresponds tonucleic acids 1 to 1725 of SEQ ID NO:7. The amino acid sequencecorresponds to amino acids 1 to 369 of SEQ ID NO:8. The coding regionwithout the 3′ untranslated region of the human DHDR-3 gene is shown inSEQ ID NO:9.

FIG. 12 depicts a structural, hydrophobicity, and antigenicity analysisof the human DHDR-3 protein.

FIG. 13 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of four“transmembrane domains” in the human DHDR-3 protein (SEQ ID NO:8).

FIGS. 14A-14B depicts the results of a search which was performedagainst the HMM database and which resulted in the identification of a“3-beta hydroxysteroid dehydrogenase domain”, a “short chaindehydrogenase domain”, and a “NAD-dependent epimerase/dehydratasedomain” in the human DHDR-3 protein. In FIG. 14A, the short chaindehydrogenase domain (SEQ ID NO:19) is aligned with DHDR-3 protein(25964; SEQ ID NO:8) residues. Also aligned is S-adenosylmethioninesynthetase domain (SEQ ID NO:22) with DHDR-3 protein residues. FIG. 14B1 depicts an alignment of 3-betahydroxysteroid dehydrogenase domain (SEQID NO:23) with DHDR-3 protein residues. FIG. 14B 2 depicts an alignmentof NAD dependent epimerase/dehydratase domain (SEQ ID NO:24) with DHDR-3protein residues.

FIG. 15 depicts the results of a search which was performed against theProDom database and which resulted in the identification of a “3-betahydroxysteroid dehydrogenase domain” in the human DHDR-3 protein (SEQ IDNO:8). The 3-beta hydroxysteroid dehydrogenase domain sequences (SEQ IDNO:25 and SEQ ID NO:26) are aligned with DHDR-3 (Subjct) proteinresidues.

FIG. 16 depicts the cDNA sequence and predicted amino acid sequence ofhuman DHDR-4 (clone Fbh21686). The nucleotide sequence corresponds tonucleic acids 1 to 1209 of SEQ ID NO:10. The amino acid sequencecorresponds to amino acids 1 to 322 of SEQ ID NO:11. The coding regionwithout the 3′ untranslated region of the human DHDR-4 gene is shown inSEQ ID NO:12.

FIG. 17 depicts an alignment of the human DHDR-4 amino acid sequence(“21686”; SEQ ID NO:11) with the amino acid sequence of Rattusnorvegicus putative short-chain dehydrogenase/reductase(“5052204_SDR_rat”; GenBank Accession Number AF099742; SEQ ID NO:13)using the CLUSTAL W (1.74) multiple sequence alignment program.Identical amino acids are indicated by stars.

FIG. 18 depicts a structural, hydrophobicity, and antigenicity analysisof the human DHDR-4 protein.

FIG. 19 depicts the results of a signal peptide prediction and a searchwhich was performed against the MEMSAT database and which resulted inthe identification of a “signal peptide” and four “transmembranedomains” in the human DHDR-4 protein (SEQ ID NO:11).

FIG. 20 depicts the results of a search which was performed against theHMM database and which resulted in the identification of a “short chaindehydrogenase domain” and a “short chain dehydrogenase/reductase domain”in the human DHDR-4 protein. The short chain dehydrogenase domain (SEQID NO:19) is aligned with DHDR-4 protein (21686; SEQ ID NO:11) residues.Also, short chain dehydrogenase/reductase C2 domain (SEQ ID NO:27) isaligned with DHDR-4 protein residues.

FIGS. 21A-21B depict the results of a search which was performed againstthe ProDom database and which resulted in the identification of a“oxidoreductase protein dehydrogenase domain” (SEQ ID NO:28, SEQ IDNO:29 and SEQ ID NO:30), a “shikimate 5-dehydrogenase domain” (SEQ IDNO:32), a “dehydrogenase domain” (SEQ ID NO:33), and a“glucose-1-dehydrogenase domain” (SEQ ID NO:31) in the human DHDR-4protein (SEQ ID NO: 11). Also depicted is an alignment of a hypotheticalprotein domain (SEQ ID NO:34 aligned with human DHDR-4 protein residues.

FIG. 22 depicts the expression levels of human DHDR-1 mRNA in varioushuman cell types and tissues, as determined by Taqman analysis. Samples:(1) normal artery; (2) normal vein; (3) aortic smooth musclecells—early; (4) coronary smooth muscle cells; (5) human microvascularendothelial cells (HMVECs)—static; (6) human microvascular endothelialcells (HMVECs)—shear; (7) normal heart; (8) heart—congestive heartfailure (CHF); (9) kidney; (10) skeletal muscle; (11) normal adiposetissue; (12) pancreas; (13) primary osteoblasts; (14) differentiatedosteoclasts; (15) normal skin; (16) normal spinal cord; (17) normalbrain cortex; (18) brain—hypothalamus; (19) nerve; (20) dorsal rootganglion (DRG); (21) resting peripheral blood mononuclear cells (PBMCs);(22) glioblastoma; (23) normal breast; (24) breast tumor; (25) normalovary; (26) ovary tumor; (27) normal prostate; (28) prostate tumor; (29)epithelial cells (prostate); (30) normal colon; (31) colon tumor; (32)normal lung; (33) lung tumor; (34) lung—chronic obstructive pulmonarydisease (COPD); (35) colon—inflammatory bowel disease (IBD); (36) normalliver; (37) liver—fibrosis; (38) dermal cells—fibroblasts; (39) normaltonsil; (40) lymph node; (41) small intestine; (42) skin—decubitus; (43)synovium; (44) bone marrow mononuclear cells (BM-MNC); (45) activatedperipheral blood mononuclear cells (PBMCs).

FIG. 23 depicts the expression levels of human DHDR-1 mRNA in varioustypes of human tumors, as determined by Taqman analysis. Samples: (1-3)normal breast; (4) breast tumor—infiltrating ductal carcinoma (IDC); (5)breast tumor—infiltrating ductal carcinoma (MD-IDC); (6-8) breasttumor—infiltrating ductal carcinoma (IDC); (9) breast tumor; (10-11)normal ovary; (12-16) ovary tumor; (17-19) normal lung; (20) lungtumor—5 mC; (21-23) lung tumor—poorly differentiated non-small cellcarcinoma of the lung (PDNSCCL); (24) lung tumor—small cell carcinoma(SCC); (25) lung tumor—AC; (26) lung tumor—ACA; (27-29) normal colon;(30-31) colon tumor—MD; (32) colon tumor; (33) colon tumor—MD-PD;(34-35) colon tumor—liver metastasis; (36) normal liver (female); (37)hemangioma; (38) human microvascular endothelial cells(HMVECs)—arrested; (39) human microvascular endothelial cells(HMVECs)—proliferating.

FIG. 24 depicts the expression levels of human DHDR-1 mRNA in varioushuman colon tumor samples, as determined by Taqman analysis. Samples:(1-6) normal colon; (7-8) adenomas; (9-15) colonic ACA-B; (16-21);colonic ACA-C; (22-27) normal liver; (28-33) colon tumor—livermetastasis; (34) colon tumor—abdominal metastasis.

FIG. 25 depicts the expression levels of human DHDR-1 mRNA in NOCsynchronized HCT116 cells at various time points after entry into thecell cycle, as determined by Taqman analysis. The time point t=0signifies the G2/M border. Samples: (1) t=0; (2) t=3; (3) t=6; (4) t=9;(5) t=15; (6) t=18; (7) t=21; (8) t=24.

FIG. 26 depicts the expression levels of human DHDR-2 mRNA in varioushuman clinical tumor samples, as determined by Taqman analysis. Samples:(1-4) normal breast; (5-11) breast tumor; (12-14) normal lung; (15-22)lung tumor; (23-25) normal colon; (26-33) colon tumor; (34-37) colontumor—liver metastasis; (38-39) normal liver; (40) normal brain; (41-43)brain tumor—glioblastoma.

FIG. 27 depicts the expression levels of human DHDR-4 mRNA in varioushuman cell types and tumors, as determined by Taqman analysis. Samples:(1) normal aorta; (2) normal fetal heart; (3) normal heart; (4)heart—congestive heart failure (CHF); (5) normal vein; (6) aortic smoothmuscle cells; (7) normal spinal cord; (8) normal brain cortex; (9)brain—hypothalamus; (10) glial cells—astrocytes; (11)brain—glioblastoma; (12) normal breast; (13) breast tumor—infiltratingductal carcinoma (IDC); (14) normal ovary; (15) ovary tumor; (16)pancreas; (17) normal prostate; (18) prostate tumor; (19) normal colon;(20) colon tumor; (21) colon—inflammatory bowel disease (IBD); (22)normal kidney; (23) normal liver; (24) liver—fibrosis; (25) normal fetalliver; (26) normal lung; (27) lung tumor; (28) lung—chronic obstructivepulmonary disease (COPD); (29) normal spleen; (30) normal tonsil; (31)normal lymph node; (32) normal thymus; (33) epithelial cells—prostate;(34) endothelial cells—aortic; (35) skeletal muscle; (36) dermalfibroblasts; (37) normal skin; (38) normal adipose tissue; (39) primaryosteoblasts; (40) undifferentiated osteoblasts; (41) differentiatedosteoblasts; (42) osteoclasts; (43) aortic smooth muscle cells(SMCs)—early; (44) aortic smooth muscle cells (SMCs)—late; (45) humanumbilical vein endothelial cells (HUVECs)—shear; (46) human umbilicalvein endothelial cells (HUVECs)—static.

FIG. 28 depicts the expression levels of human DHDR-4 mRNA in variouscell types and tissues, as determined by Taqman analysis. Samples: (1-2)normal liver; (3-4) HBV+liver; (5) HCV+liver; (6) HepG2-B cells; (7)HepG2.2.15-B cells; (8) HepG2 cells (no treatment); (9) HepG2cells—treated with Bayer compound (IC50); (10) HepG2 cells—treated withBayer compound (IC100); (11) HepG2.2.15 cells (no treatment); (12)HepG2.2.15 cells—treated with Bayer compound (IC50); (13) HepG2.2.15cells—treated with Bayer compound (IC100); (14) HepG2 control; (15)HepG2 cells transfected with the HBV-X gene; (16) HuH7 cells; (17-19)ganglia; (20) NT2/KOS—0 hr.; (21) NT2/KOS—2.5 hr.; (22) NT2/KOS—5 hr.;(23) NT2/KOS—7 hr.; (24) MRC/VZV—mock; (25) MRC/VZV—18 hr.; (26)MRC/VZV—72 hr.

FIG. 29 depicts the expression levels of human DHDR-4 mRNA in varioushuman tumor samples, as determined by Taqman analysis. Samples: (1-4)normal colon; (5-11) colon tumor; (12-15) colon tumor—liver metastasis;(16-17) normal liver; (18-21) normal brain; (22-27) braintumor—glioblastoma; (28-29) human microvascular endothelial cells(HMVECs); (30) placenta; (31-32) fetal adrenal gland; (33-34) fetalliver.

FIG. 30 depicts the expression levels of human DHDR-4 mRNA in varioushuman tumors and synchronized A549 cells at various time points afterentry into the cell cycle, as determined by Taqman analysis. The timepoint t=0 signifies the G2/M border. Samples: (1-4) normal breast;(5-10) breast tumor; (11-14) normal ovary; (15-22) ovary tumor; (23-26)normal lung; (27-34) lung tumor; (35-42) synchronized A549 cells: (35)t=0; (36) t=3 (RT); (37) t=3 (RT); (38) t=6; (39) t=9; (40) t=12 (RT);(41) t=18; (42) t=24.

FIGS. 31A-31B depict the cDNA sequence and predicted amino acid sequenceof mouse DHDR-2 (clone m21481). The nucleotide sequence, correspondingto nucleic acids 1 to 1108 of SEQ ID NO:14, is shown in FIG. 31A. Thecoding region, corresponding to SEQ ID NO:16, is underlined. The aminoacid sequence, corresponding to amino acids 1 to 311 of SEQ ID NO:15, isshown in FIG. 31B.

FIGS. 32A-32B depict an alignment of the mouse DHDR-2 nucleotidesequence (“M21484”; SEQ ID NO:14) with the human DHDR-2 nucleotidesequence (“h21484”; SEQ ID NO:4) using the GAP program in the GCGsoftware package (nwsgapdna.cmp matrix) and a gap weight of 12 and alength weight of 4. As shown in the alignment, the mouse and humanDHDR-2 nucleotide sequences are about 88.1% identical.

FIG. 33 depicts an alignment of the mouse DHDR-2 amino acid sequence(“m21484”; SEQ ID NO:15) with the human DHDR-2 amino acid sequence(“h21484”; SEQ ID NO:5) using the GAP program in the GCG softwarepackage (Blosum 62 matrix) and a gap weight of 12 and a length weight of4. As shown in the alignment, the mouse and human DHDR-2 amino acidsequences are about 91.3% identical.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “dehydrogenase” or “DHDR” nucleicacid and protein molecules, which are novel members of a family ofenzymes possessing dehydrogenase activity. These novel molecules arecapable of oxidizing or reducing biological molecules by catalyzing thetransfer of a hydride moiety and, thus, play a role in or function in avariety of cellular processes, e.g., proliferation, growth,differentiation, migration, immune responses, hormonal responses, inter-or intra-cellular communication, and viral infection.

As used herein, the term “dehydrogenase” includes a molecule which isinvolved in the oxidation or reduction of a biochemical molecule (e.g.,an amino acid, a vitamin, a steroid such as a glucocorticoid, or anucleic acid), by catalyzing the transfer of a hydride ion to or fromthe biochemical molecule. Dehydrogenase molecules are involved in themetabolism and catabolism of biochemical molecules necessary for energyproduction or storage, for intra- or inter-cellular signaling, formetabolism or catabolism of metabolically important biomolecules, andfor detoxification of potentially harmful compounds. Examples ofdehydrogenases include alcohol dehydrogenases, aldehyde dehydrogenases,steroid dehydrogenases, and lipid dehydrogenases. Thus, the DHDRmolecules of the present invention provide novel diagnostic targets andtherapeutic agents to control dehydrogenase-associated disorders.

As used herein, a “dehydrogenase-associated disorder” includes adisorder, disease or condition which is caused or characterized by amisregulation (e.g., downregulation or upregulation) of dehydrogenaseactivity. Dehydrogenase-associated disorders can detrimentally affectcellular functions such as cellular proliferation, growth,differentiation, or migration, inter- or intra-cellular communication;tissue function, such as cardiac function or musculoskeletal function;systemic responses in an organism, such as nervous system responses,hormonal responses (e.g., insulin response), susceptibility topathogenic infections (e.g., viral infections), or immune responses; andprotection of cells from toxic compounds (e.g., carcinogens, toxins, ormutagens). Examples of dehydrogenase-associated disorders include CNSdisorders such as cognitive and neurodegenerative disorders, examples ofwhich include, but are not limited to, Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of dehydrogenase-associated disorders includecardiac-related disorders. Cardiovascular system disorders in which theDHDR molecules of the invention may be directly or indirectly involvedinclude arteriosclerosis, ischemia reperfusion injury, restenosis,arterial inflammation, vascular wall remodeling, ventricular remodeling,rapid ventricular pacing, coronary microembolism, tachycardia,bradycardia, pressure overload, aortic bending, coronary arteryligation, vascular heart disease, atrial fibrilation, Jervell syndrome,Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure,sinus node dysfunction, angina, heart failure, hypertension, atrialfibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. DHDR-mediated or related disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

Dehydrogenase associated disorders also include cellular proliferation,growth, differentiation, or migration disorders. Cellular proliferation,growth, differentiation, or migration disorders include those disordersthat affect cell proliferation, growth, differentiation, or migrationprocesses. As used herein, a “cellular proliferation, growth,differentiation, or migration process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The DHDR molecules of the present invention areinvolved in signal transduction mechanisms, which are known to beinvolved in cellular growth, differentiation, and migration processes.Thus, the DHDR molecules may modulate cellular growth, differentiation,or migration, and may play a role in disorders characterized byaberrantly regulated growth, differentiation, or migration. Suchdisorders include cancer, e.g., carcinomas, sarcomas, leukemias, andlymphomas; tumor angiogenesis and metastasis; skeletal dysplasia;hepatic disorders; and hematopoietic and/or myeloproliferativedisorders.

DHDR-associated or related disorders also include hormonal disorders,such as conditions or diseases in which the production and/or regulationof hormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

DHDR-associated or related disorders also include immune disorders, suchas autoimmune disorders or immune deficiency disorders, e.g., allergies,transplant rejection, responses to pathogenic infection (e.g.,bacterial, viral, or parasitic infection), lupus, multiple sclerosis,congenital X-linked infantile hypogammaglobulinemia, transienthypogammaglobulinemia, common variable immunodeficiency, selective IgAdeficiency, chronic mucocutaneous candidiasis, or severe combinedimmunodeficiency.

DHDR-associated or related disorders also include viral disorders, i.e.,disorders affected or caused by infection by viruses (e.g., hepatitis A,hepatitis B, hepatitis delta, and other hepadnaviruses; Coxsackie Bviruses; Epstein-Barr virus; adenovirus; rhinoviruses; humanimmunodeficiency virus (HIV); vaccinia virus; human T cell leukemiavirus; RD114 virus; herpes simplex, herpes zoster, and otherherpesviruses; Marek's disease virus; Yamaguchi sarcoma virus; humanpapillomaviruses; poliovirus; poxviruses; influenza virus;cytomegalovirus; encephalitis viruses; measles viruses; and ebola andother hemorrhagic viruses). Such disorders include, but are not limitedto, hepatocellularcarcinoma, cirrhosis of the liver, cervical carcinoma,Burkitt's lymphoma, lymphoproliferative disease, Kaposi's sarcoma, Tcell leukemia, B cell lymphoma, plasmablastic lymphoma, Rasmussen'ssyndrome, Marek's disease, warts (including common, genital, and plantarwarts), genital herpes, common colds, acquired immune deficiencysyndrome (AIDS), polymyositis, immunorestitution disease, chicken pox,shingles, ebola and other hemorrhagic fever diseases, cold sores,transient or acute hepatitis, chronic hepatitis, influenza, Reyesyndrome, measles, Paget's disease, viral encephalitis, viral pneumonia,and viral meningitis.

Viral disorders also include disorders or conditions influenced by virusor viral activity. As used interchangeably herein, the terms “viralactivity” and “virus activity” includes any activity known in the art tobe characteristic of a virus and which can be detected by methods knownin the art. For example, viral activity includes, but it not limited to,the ability of a virus to infect a cell or an organism (e.g., a human),the ability of a virus to replicate or reproduce in a cell or anorganism (e.g., a human), the ability of a virus to induce an immuneresponse in vitro or in vivo, the ability to induce expression of viralgenes, and/or the ability to induce expression of host or exogenousgenes not normally expressed by an uninfected cell.

DHDR-associated or related disorders also include disorders affectingtissues in which DHDR protein is expressed, e.g., liver, hepatocytes,hepatitis B-infected hepatocytes, HepG2 cells, hepatitis B-infectedHepG2.2.15 cells, kidney, brain, primary osteoblasts, pituitary, CaCOcells, keratinocytes, aortic endothelial cells, fetal kidney, fetallung, mammary epithelium, fetal spleen, fetal liver, umbilical smoothmuscle, RAII Burkitt Lymphoma cells, lung, prostate, K53 red bloodcells, fetal dorsal spinal cord, insulinoma cells, normal breast andovarian epithelia, retina, HMC-1 mast cells, ovarian ascites, d8dendritic cells, megakaryocytes, human mobilized bone morrow, mammarycarcinoma, melanoma cells, lymph, vein, U937/A70p B cells, A549concells, WT LN Cap testosterone cells, and esophagus.

As used herein, a “dehydrogenase-mediated activity” includes an activitywhich involves the oxidation or reduction of one or more biochemicalmolecules, e.g., biochemical molecules (e.g., glucocorticoids) in aneuronal cell, a liver cell, or a tumor cell associated with theregulation of one or more cellular processes. Dehydrogenase-mediatedactivities include the oxidation or reduction of biochemical moleculesnecessary for energy production or storage, for intra- or inter-cellularsignaling, for metabolism or catabolism of metabolically importantbiomolecules, for viral infection, and for detoxification of potentiallyharmful compounds.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., monkey proteins. Members of afamily may also have common functional characteristics.

For example, the family of DHDR proteins comprises at least one“transmembrane domain”. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example, Zagotta W. N. et al., (1996) Annu. Rev.Neurosci. 19:235-263, the contents of which are incorporated herein byreference. Amino acid residues 159-175 of the native DHDR-1 protein arepredicted to comprise a transmembrane domain (see FIG. 3). Amino acidresidues 7-23 of the native DHDR-2 protein and residues 265-283 of themature DHDR-2 protein are predicted to comprise a transmembrane domain(see FIG. 8). Amino acid residues 10-26, 73-90, 289-305, and 312-333 ofthe native DHDR-3 protein are predicted to comprise transmembranedomains (see FIG. 13). Amino acid residues 29-50, 170-188, 108-224, and258-275 of the native DHDR-4 protein and residues 10-31, 151-169,189-205, and 239-256 of the mature DHDR-4 protein are predicted tocomprise transmembrane domains (see FIG. 19). Accordingly, DHDR proteinshaving at least 50-60% homology, preferably about 60-70%, morepreferably about 70-80%, or about 80-90% homology with a transmembranedomain of human DHDR are within the scope of the invention.

In another embodiment of the invention, a DHDR protein of the presentinvention is identified based on the presence of a signal peptide. Theprediction of such a signal peptide can be made, for example, utilizingthe computer algorithm SignalP (Henrik et al. (1997) Prot. Eng. 10:1-6). As used herein, a “signal sequence” or “signal peptide” includes apeptide containing about 15 or more amino acids which occurs at theN-terminus of secretory and membrane bound proteins and which contains alarge number of hydrophobic amino acid residues. For example, a signalsequence contains at least about 10-30 amino acid residues, preferablyabout 15-25 amino acid residues, more preferably about 18-20 amino acidresidues, and more preferably about 19 amino acid residues, and has atleast about 35-65%, preferably about 38-50%, and more preferably about40-45% hydrophobic amino acid residues (e.g., Valine, Leucine,Isoleucine or Phenylalanine). Such a “signal sequence”, also referred toin the art as a “signal peptide”, serves to direct a protein containingsuch a sequence to a lipid bilayer, and is cleaved in secreted andmembrane bound proteins. A signal sequence was identified in the aminoacid sequence of human DHDR-2 at about amino acids 1-18 of SEQ ID NO:5.A signal sequence was also identified in the amino acid sequence ofhuman DHDR-4 at about amino acids 1-19 of SEQ ID NO:11.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of an “aldehyde dehydrogenase familydomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “aldehyde dehydrogenase family domain” includes aprotein domain having an amino acid sequence of about 350-550 amino acidresidues and a bit score of at least 149.8. Preferably, an aldehydedehydrogenase family domain includes at least about 400-500, or morepreferably about 448 amino acid residues, and a bit score of about 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, or 250 or more. To identify the presence of analdehyde dehydrogenase family domain in a DHDR protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein is searched against a database ofknown protein domains (e.g., the HMM database). The aldehydedehydrogenase family domain (HMM) has been assigned the PFAM AccessionPF00171 (see the Pfam website, available online through WashingtonUniversity in Saint Louis). A search was performed against the HMMdatabase resulting in the identification of an aldehyde dehydrogenasefamily domain in the amino acid sequence of human DHDR-1 (SEQ ID NO:2)at about residues 47-494 of SEQ ID NO:2. The results of the search areset forth in FIG. 4.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of an “aldehyde dehydrogenaseoxidoreductase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “aldehyde dehydrogenaseoxidoreductase domain” includes a protein domain having an amino acidsequence of about 550-750 amino acid residues and having a bit score forthe alignment of the sequence to the aldehyde dehydrogenaseoxidoreductase domain of at least 280. Preferably, an aldehydedehydrogenase oxidoreductase domain includes at least about 600-700, ormore preferably about 670 amino acid residues, and has a bit score forthe alignment of the sequence to the aldehyde dehydrogenaseoxidoreductase domain of at least 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400 or higher. The aldehyde dehydrogenaseoxidoreductase domain has been assigned ProDom entry 135. To identifythe presence of an aldehyde dehydrogenase oxidoreductase domain in aDHDR protein, and to make the determination that a protein of interesthas a particular profile, the amino acid sequence of the protein issearched against a database of known protein domains (e.g., the ProDomdatabase) using the default parameters (available online through theProDom website). A search was performed against the ProDom databaseresulting in the identification of an aldehyde dehydrogenaseoxidoreductase domain in the amino acid sequence of human DHDR-1 (SEQ IDNO:2) at about residues 101-770 of SEQ ID NO:2. The results of thesearch are set forth in FIGS. 5A-5B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “short chain dehydrogenase domain”in the protein or corresponding nucleic acid molecule. As used herein,the term “short chain dehydrogenase domain” includes a protein domainhaving an amino acid sequence of about 100-300 amino acid residues, anda bit score of at least 120.0-162.5. Preferably, a short chaindehydrogenase domain includes at least about 150-250, or more preferablyabout 187-195 amino acid residues, and has a bit score of at least 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, or more. To identify the presence of a short chaindehydrogenase domain in a DHDR protein, and make the determination thata protein of interest has a particular profile, the amino acid sequenceof the protein is searched against a database of known protein domains(e.g., the HMM database). The short chain dehydrogenase domain (HMM) hasbeen assigned the PFAM Accession PF00106 (see the Pfam website,available online through Washington University in Saint Louis). A searchwas performed against the HMM database resulting in the identificationof a short chain dehydrogenase domain in the amino acid sequence ofhuman DHDR-2 (SEQ ID NO:5) at about residues 38-227 of SEQ ID NO:5. Theresults of the search are set forth in FIG. 9. A search was alsoperformed against the HMM database resulting in the identification of ashort chain dehydrogenase domain in the amino acid sequence of humanDHDR-3 (SEQ ID NO:8) at about residues 10-197 of SEQ ID NO:8. Theresults of this search are set forth in FIGS. 14A-14B. Another searchperformed against the HMM database resulted in the identification of ashort chain dehydrogenase domain in the amino acid sequence of humanDHDR-4 (SEQ ID NO:11) at about residues 38-226 of SEQ ID NO:11. Theresults of this search are set forth in FIG. 20.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of an “oxidoreductase proteindehydrogenase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “oxidoreductase protein dehydrogenasedomain” includes a protein domain having an amino acid sequence of about50-300 amino acid residues and having a bit score for the alignment ofthe sequence to the oxidoreductase protein dehydrogenase domain of atleast 113. Preferably, an oxidoreductase protein dehydrogenase domainincludes at least about 100-250, or more preferably about 120-200 aminoacid residues, and has a bit score for the alignment of the sequence tothe oxidoreductase protein dehydrogenase domain of at least 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or higher. Theoxidoreductase protein dehydrogenase domain has been assigned ProDomentry 11. To identify the presence of an oxidoreductase proteindehydrogenase domain in a DHDR protein, and to make the determinationthat a protein of interest has a particular profile, the amino acidsequence of the protein is searched against a database of known proteindomains (e.g., the ProDom database) using the default parameters(available online through the ProDom website). A search was performedagainst the ProDom database resulting in the identification of anoxidoreductase protein dehydrogenase domain in the amino acid sequenceof human DHDR-2 (SEQ ID NO:5) at about residues 99-219 of SEQ ID NO:5.The results of the search are set forth in FIG. 10. Another search wasperformed against the ProDom database, resulting in the identificationof an oxidoreductase protein dehydrogenase domain in the amino acidsequence of human DHDR-4 (SEQ ID NO:11) at about residues 37-231 of SEQID NO:11. The results of this search are set forth in FIGS. 21A-21B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of an “NAD-dependentepimerase/dehydratase domain” in the protein or corresponding nucleicacid molecule. As used herein, the term “NAD-dependentepimerase/dehydratase domain” includes a protein domain having an aminoacid sequence of about 250-450 amino acid residues. Preferably, anNAD-dependent epimerase/dehydratase domain includes at least about300-400, or more preferably about 354 amino acid residues. To identifythe presence of an NAD-dependent epimerase/dehydratase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the HMM database).The NAD-dependent epimerase/dehydratase domain (HMM) has been assignedthe PFAM Accession PF01370 (see the Pfam website, available onlinethrough Washington University in Saint Louis). A search was performedagainst the HMM database resulting in the identification of anNAD-dependent epimerase/dehydratase domain in the amino acid sequence ofhuman DHDR-3 (SEQ ID NO:8) at about residues 12-365 of SEQ ID NO:8. Theresults of the search are set forth in FIGS. 14A-14B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “3-beta hydroxysteroiddehydrogenase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “3-beta hydroxysteroid dehydrogenasedomain” includes a protein domain having an amino acid sequence of about250-450 amino acid residues and having a bit score for the alignment ofthe sequence to the 3-beta hydroxysteroid dehydrogenase domain of atleast 395-676.9. Preferably, a 3-beta hydroxysteroid dehydrogenasedomain includes at least about 300-400, or more preferably about 352-365amino acid residues, and has a bit score for the alignment of thesequence to the 3-beta hydroxysteroid dehydrogenase domain of at least300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, 725, 750, 775, 800, 825 or higher. The 3-betahydroxysteroid dehydrogenase domain has been assigned ProDom entry 1280.To identify the presence of a 3-beta hydroxysteroid dehydrogenase domainin a DHDR protein, and to make the determination that a protein ofinterest has a particular profile, the amino acid sequence of theprotein is searched against a database of known protein domains (e.g.,the ProDom database) using the default parameters (available onlinethrough the ProDom website). A search was performed against the ProDomdatabase resulting in the identification of a 3-beta hydroxysteroiddehydrogenase domain in the amino acid sequence of human DHDR-3 (SEQ IDNO:8) at about residues 11-362 of SEQ ID NO:8. The results of the searchare set forth in FIG. 15. A search was also performed against the HMMdatabase resulting in the identification of a 3-beta hydroxysteroiddehydrogenase domain (PFAM accession PF01073, see the Pfam website,available online through Washington University in Saint Louis) in theamino acid sequence of human DHDR-3 (SEQ ID NO:8) at about residues1-365 of SEQ ID NO:8. The results of the search are set forth in FIGS.14A-14B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “short-chaindehydrogenase/reductase domain” in the protein or corresponding nucleicacid molecule. As used herein, the term “short-chaindehydrogenase/reductase domain” includes a protein domain having anamino acid sequence of about 10-100 amino acid residues, and a bit scoreof at least 47.2. Preferably, a short-chain dehydrogenase/reductasedomain includes at least about 20-75, or more preferably about 31 aminoacid residues, and has a bit score of at least 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more. To identifythe presence of a short-chain dehydrogenase/reductase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the HMM database).The short-chain dehydrogenase/reductase domain (HMM) has been assignedthe PFAM Accession PF00678 (see the Pfam website, available onlinethrough Washington University in Saint Louis). A search was performedagainst the HMM database resulting in the identification of ashort-chain dehydrogenase/reductase domain in the amino acid sequence ofhuman DHDR-4 (SEQ ID NO:11) at about residues 250-280 of SEQ ID NO:11.The results of the search are set forth in FIG. 20.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “shikimate 5-dehydrogenase domain”in the protein or corresponding nucleic acid molecule. As used herein,the term “shikimate 5-dehydrogenase domain” includes a protein domainhaving an amino acid sequence of about 10-100 amino acid residues andhaving a bit score for the alignment of the sequence to the shikimate5-dehydrogenase domain of at least 86. Preferably, a shikimate5-dehydrogenase domain includes at least about 25-75, or more preferablyabout 48 amino acid residues, and has a bit score for the alignment ofthe sequence to the shikimate 5-dehydrogenase domain of at least 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher. The shikimate5-dehydrogenase domain has been assigned ProDom entry 95301. To identifythe presence of a shikimate 5-dehydrogenase domain in a DHDR protein,and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the ProDom database)using the default parameters (available online through the ProDomwebsite). A search was performed against the ProDom database resultingin the identification of a shikimate 5-dehydrogenase domain in the aminoacid sequence of human DHDR-4 (SEQ ID NO:11) at about residues 35-82 ofSEQ ID NO:11. The results of the search are set forth in FIGS. 21A-21B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “dehydrogenase domain” in theprotein or corresponding nucleic acid molecule. As used herein, the term“dehydrogenase domain” includes a protein domain having an amino acidsequence of about 10-100 amino acid residues and having a bit score forthe alignment of the sequence to the dehydrogenase domain of at least84. Preferably, a dehydrogenase domain includes at least about 25-75, ormore preferably about 50 amino acid residues, and has a bit score forthe alignment of the sequence to the dehydrogenase domain of at least20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher. Thedehydrogenase domain has been assigned ProDom entry 73753. To identifythe presence of a dehydrogenase domain in a DHDR protein, and to makethe determination that a protein of interest has a particular profile,the amino acid sequence of the protein is searched against a database ofknown protein domains (e.g., the ProDom database) using the defaultparameters (available online through the ProDom website). A search wasperformed against the ProDom database resulting in the identification ofa dehydrogenase domain in the amino acid sequence of human DHDR-4 (SEQID NO:11) at about residues 237-286 of SEQ ID NO:11. The results of thesearch are set forth in FIGS. 21A-21B.

In another embodiment, a DHDR molecule of the present invention isidentified based on the presence of a “glucose-1-dehydrogenase domain”in the protein or corresponding nucleic acid molecule. As used herein,the term “glucose-1-dehydrogenase domain” includes a protein domainhaving an amino acid sequence of about 10-100 amino acid residues andhaving a bit score for the alignment of the sequence to theglucose-1-dehydrogenase domain of at least 92. Preferably, adehydrogenase domain includes at least about 25-75, or more preferablyabout 45 amino acid residues, and has a bit score for the alignment ofthe sequence to the dehydrogenase domain of at least 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or higher. Theglucose-1-dehydrogenase domain has been assigned ProDom entry 77223. Toidentify the presence of a glucose-1-dehydrogenase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the ProDom database)using the default parameters (available online through the ProDomwebsite). A search was performed against the ProDom database resultingin the identification of a dehydrogenase domain in the amino acidsequence of human DHDR-4 (SEQ ID NO:11) at about residues 243-287 of SEQID NO:11. The results of the search are set forth in FIGS. 21A-21B.

In a preferred embodiment, the DHDR molecules of the invention includeat least one or more of the following domains: a transmembrane domain, asignal peptide domain, an aldehyde dehydrogenase oxidoreductase domain,an aldehyde dehydrogenase family domain, a short chain dehydrogenasedomain, an oxidoreductase protein dehydrogenase domain, a 3-betahydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain, anda glucose-1-dehydrogenase domain.

Isolated proteins of the present invention, preferably DHDR proteins,have an amino acid sequence sufficiently identical to the amino acidsequence of SEQ ID NO:2, 5, 8, 11, or 15, or are encoded by a nucleotidesequence sufficiently identical to SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12,14, or 16. As used herein, the term “sufficiently identical” refers to afirst amino acid or nucleotide sequence which contains a sufficient orminimum number of identical or equivalent (e.g., an amino acid residuewhich has a similar side chain) amino acid residues or nucleotides to asecond amino acid or nucleotide sequence such that the first and secondamino acid or nucleotide sequences share common structural domains ormotifs and/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains have at least30%, 40%, or 50% homology, preferably 60% homology, more preferably70%-80%, and even more preferably 90-95% homology across the amino acidsequences of the domains and contain at least one and preferably twostructural domains or motifs, are defined herein as sufficientlyidentical. Furthermore, amino acid or nucleotide sequences which shareat least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or90-95% homology and share a common functional activity are definedherein as sufficiently identical.

As used interchangeably herein, an “DHDR activity”, “biological activityof DHDR” or “functional activity of DHDR”, refers to an activity exertedby a DHDR protein, polypeptide or nucleic acid molecule on a DHDRresponsive cell or tissue, or on a DHDR protein substrate, as determinedin vivo, or in vitro, according to standard techniques. In oneembodiment, a DHDR activity is a direct activity, such as an associationwith a DHDR-target molecule. As used herein, a “target molecule” or“binding partner” is a molecule with which a DHDR protein binds orinteracts in nature, such that DHDR-mediated function is achieved. ADHDR target molecule can be a non-DHDR molecule or a DHDR protein orpolypeptide of the present invention (e.g., NAD+, NADP+, or othercofactor). In an exemplary embodiment, a DHDR target molecule is a DHDRligand (e.g., an alcohol, an aldehyde, a lipid, or a steroid (e.g., aglucocorticoid)). Alternatively, a DHDR activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the DHDR protein with a DHDR ligand. The biological activities ofDHDR are described herein. For example, the DHDR proteins of the presentinvention can have one or more of the following activities: 1) modulatemetabolism and catabolism of biochemical-molecules necessary for energyproduction or storage, 2) modulate intra- or inter-cellular signaling,3) modulate metabolism or catabolism of metabolically importantbiomolecules (e.g., glucocorticoids), 4) modulate detoxification ofpotentially harmful compounds, 5) modulate viral infection (e.g., bymodulating viral gene expression), 6) act as a transcriptional cofactorfor viral gene activation, 7) modulate viral activity, and/or 8)modulate cellular proliferation.

Accordingly, another embodiment of the invention features isolated DHDRproteins and polypeptides having a DHDR activity. Other preferredproteins are DHDR proteins having one or more of the following domains:a transmembrane domain, a signal peptide domain, an aldehydedehydrogenase oxidoreductase domain, an aldehyde dehydrogenase familydomain, a short chain dehydrogenase domain, an oxidoreductase proteindehydrogenase domain, a 3-beta hydroxysteroid dehydrogenase domain, aNAD-dependent epimerase/dehydratase domain, a short chaindehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain, adehydrogenase domain, or a glucose-1-dehydrogenase domain and,preferably, a DHDR activity.

Additional preferred proteins have at least one transmembrane domain,and one or more of a signal peptide domain, an aldehyde dehydrogenaseoxidoreductase domain, an aldehyde dehydrogenase family domain, a shortchain dehydrogenase domain, an oxidoreductase protein dehydrogenasedomain, a 3-beta hydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain, or aglucose-1-dehydrogenase domain, and are, preferably, encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a complement of a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4,6, 7, 9, 10, 12, 14, or 16.

The nucleotide sequence of the isolated human DHDR-1 cDNA and thepredicted amino acid sequence of the human DHDR-1 polypeptide are shownin FIGS. 1A-1D and in SEQ ID NOs:1 and 2, respectively. The nucleotidesequence of the isolated human DHDR-2 cDNA and the predicted amino acidsequence of the human DHDR-2 polypeptide are shown in FIGS. 6A-6B and inSEQ ID NOs:4 and 5, respectively. The nucleotide sequence of theisolated human DHDR-3 cDNA and the predicted amino acid sequence of thehuman DHDR-3 polypeptide are shown in FIGS. 11A-11B and in SEQ ID NOs:7and 8, respectively. The nucleotide sequence of the isolated humanDHDR-4 cDNA and the predicted amino acid sequence of the human DHDR-4polypeptide are shown in FIG. 16 and in SEQ ID NOs:10 and 11,respectively. The nucleotide sequence of the isolated mouse DHDR-2 andthe predicted amino acid sequence of the mouse DHDR-2 polypeptide areshown in FIGS. 31A and 31B, respectively, and in SEQ ID NOs:14 and 15,respectively. Plasmids containing the nucleotide sequence encoding humanDHDR-2, and DHDR-4, and mouse DHDR-2 were deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, on May 9, 2000, and Mar. 22, 2001, respectively, andassigned Accession Numbers PTA-1845, and ______, respectively. Thesedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thatdeposits are required under 35 U.S.C. §112.

The human DHDR-1 gene, which is approximately 2660 nucleotides inlength, encodes a protein having a molecular weight of approximately88.0 kD and which is approximately 802 amino acid residues in length.The human DHDR-2 gene, which is approximately 1379 nucleotides inlength, encodes a protein having a molecular weight of approximately34.2 kD and which is approximately 311 amino acid residues in length.The human DHDR-3 gene, which is approximately 1725 nucleotides inlength, encodes a protein having a molecular weight of approximately40.5 kD and which is approximately 369 amino acid residues in length.The human DHDR-4 gene, which is approximately 1209 nucleotides inlength, encodes a protein having a molecular weight of approximately35.4 kD and which is approximately 322 amino acid residues in length.The mouse DHDR-2 gene, which is approximately 1108 nucleotides inlength, encodes a protein having a molecular weight of approximately34.2 kD and which is approximately 311 amino acid residues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode DHDR proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify DHDR-encoding nucleic acid molecules (e.g., DHDRmRNA) and fragments for use as PCR primers for the amplification ormutation of DHDR nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated DHDR nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9,10, 12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or portion of thenucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16,or the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number PTA-1845, as a hybridization probe, DHDRnucleic acid molecules can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession NumberPTA-1845 can be isolated by the polymerase chain reaction (PCR) usingsynthetic oligonucleotide primers designed based upon the sequence ofSEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number PTA-1845.

A nucleic acid of the invention can be amplified using cDNA, mRNA or,alternatively, genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to DHDR nucleotide sequences can be prepared by standardsynthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, 3, 4,6, 7, 9, 10, 12, 14, or 16. This cDNA may comprise sequences encodingthe human DHDR-1 protein (i.e., “the coding region”, from nucleotides63-2468), as well as 5′ untranslated sequences (nucleotides 1-62) and 3′untranslated sequences (nucleotides 2469-2660) of SEQ ID NO:1. This cDNAmay comprise sequences encoding the human DHDR-2 protein (i.e., “thecoding region”, from nucleotides 331-1263), as well as 5′ untranslatedsequences (nucleotides 1-330) and 3′ untranslated sequences (nucleotides1264-1379) of SEQ ID NO:4. This cDNA may comprise sequences encoding thehuman DHDR-3 protein (i.e., “the coding region”, from nucleotides281-1387), as well as 5′ untranslated sequences (nucleotides 1-280) and3′ untranslated sequences (nucleotides 1388-1725) of SEQ ID NO:7. ThiscDNA may comprise sequences encoding the human DHDR-4 protein (i.e.,“the coding region”, from nucleotides 61-1026), as well as 5′untranslated sequences (nucleotides 1-60) and 3′ untranslated sequences(nucleotides 1027-1209) of SEQ ID NO:10. This cDNA may comprisesequences encoding the mouse DHDR-2 protein (i.e., “the coding region”,from nucleotides 102-1034), as well as 5′ untranslated sequences(nucleotides 1-101) and 3′ untranslated sequences (nucleotides1035-1108) of SEQ ID NO:14. Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:1 (e.g., nucleotides63-2468, corresponding to SEQ ID NO:3), only the coding region of SEQ IDNO:4 (e.g., nucleotides 331-1263, corresponding to SEQ ID NO:6), onlythe coding region of SEQ ID NO:7 (e.g., nucleotides 281-1387,corresponding to SEQ ID NO:9), only the coding region of SEQ ID NO:10(e.g., nucleotides 61-1026, corresponding to SEQ ID NO:12), or only thecoding region of SEQ ID NO:14 (e.g., nucleotides 102-1034, correspondingto SEQ ID NO:16).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14,or 16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number PTA-1845 or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845 is onewhich is sufficiently complementary to the nucleotide sequence shown inSEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number PTA-1845 such that it can hybridize to the nucleotidesequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number PTA-1845 respectively, thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, 99.99% or more identical to the entire length of the nucleotidesequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or theentire length of the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845, or a portionof any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10,12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845, for example, afragment which can be used as a probe or primer or a fragment encoding aportion of a DHDR protein, e.g., a biologically active portion of a DHDRprotein. The nucleotide sequences determined from the cloning of theDHDR-1, DHDR-2, DHDR-3, and DHDR-4 genes allow for the generation ofprobes and primers designed for use in identifying and/or cloning otherDHDR family members, as well as DHDR homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ IDNO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession NumberPTA-1845, of an anti-sense sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10,12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845, or of anaturally occurring allelic variant or mutant of SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845. In oneembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is greater than 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900,1900-1950, 1950-2000 or more nucleotides in length and hybridizes understringent hybridization conditions to a nucleic acid molecule of SEQ IDNO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession NumberPTA-1845.

Probes based on the DHDR nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a DHDR protein, such as by measuring a level ofa DHDR-encoding nucleic acid in a sample of cells from a subject e.g.,detecting DHDR mRNA levels or determining whether a genomic DHDR genehas been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aDHDR protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number PTA-1845, which encodes a polypeptide having a DHDRbiological activity (the biological activities of the DHDR proteins aredescribed herein), expressing the encoded portion of the DHDR protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the DHDR protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10,12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845, due todegeneracy of the genetic code and thus encode the same DHDR proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequenceshown in SEQ ID NO:2, 5, 8, 11, or 15.

In addition to the DHDR nucleotide sequences shown in SEQ ID NO:1, 3, 4,6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number PTA-1845, it willbe appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theDHDR proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the DHDR genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a DHDRprotein, preferably a mammalian DHDR protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of DHDR include both functional and non-functional DHDRproteins. Functional allelic variants are naturally occurring amino acidsequence variants of the human DHDR protein that maintain the ability tobind a DHDR ligand or substrate and/or modulate cell proliferationand/or migration mechanisms. Functional allelic variants will typicallycontain only conservative substitution of one or more amino acids of SEQID NO:2, 5, 8, 11, or 15, or substitution, deletion or insertion ofnon-critical residues in non-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human DHDR protein that do not have the abilityto either bind a DHDR ligand and/or modulate any of the DHDR activitiesdescribed herein. Non-functional allelic variants will typically containa non-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15,or a substitution, insertion or deletion in critical residues orcritical regions of the protein.

The present invention further provides non-human orthologues of thehuman DHDR proteins. Orthologues of the human DHDR protein are proteinsthat are isolated from non-human organisms and possess the same DHDRligand binding and/or modulation of membrane excitability activities ofthe human DHDR protein. Orthologues of the human DHDR protein canreadily be identified as comprising an amino acid sequence that issubstantially identical to SEQ ID NO:2, 5, 8, or 11 (e.g., the mouseDHDR-2 of SEQ ID NO:15).

Moreover, nucleic acid molecules encoding other DHDR family members and,thus, which have a nucleotide sequence which differs from the DHDRsequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number PTA-1845 are intended to be within the scope of theinvention. For example, another DHDR cDNA can be identified based on thenucleotide sequence of human or mouse DHDR. Moreover, nucleic acidmolecules encoding DHDR proteins from different species, and which,thus, have a nucleotide sequence which differs from the DHDR sequencesof SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number PTA-1845 are intended to be within the scope of theinvention. For example, a mouse DHDR cDNA can be identified based on thenucleotide sequence of a human DHDR.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the DHDR cDNAs of the invention can be isolated based ontheir homology to the DHDR nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the DHDR cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the DHDR gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10,12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number PTA-1845. In otherembodiment, the nucleic acid is at least 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900,1900-1950, 1950-2000 or more nucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995), oralternatively 0.2×SSC, 1% SDS.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 3,4, 6, 7, 9, 10, 12, 14, or 16, and corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the DHDRsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14 or 16, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number PTA-1845, thereby leading to changes in theamino acid sequence of the encoded DHDR proteins, without altering thefunctional ability of the DHDR proteins. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of DHDR (e.g., the sequence of SEQ ID NO:2, 5, 8,11, or 15) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the DHDR proteinsof the present invention, e.g., those present in a transmembrane domain,are predicted to be particularly unamenable to alteration. Furthermore,additional amino acid residues that are conserved between the DHDRproteins of the present invention and other members of the DHDR familyare not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding DHDR proteins that contain changes in amino acidresidues that are not essential for activity. Such DHDR proteins differin amino acid sequence from SEQ ID NO:2, 5, 8, 11, or 15, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 92%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to SEQ ID NO:2, 5, 8, 11, or 15.

An isolated nucleic acid molecule encoding a DHDR protein identical tothe protein of SEQ ID NO:2, 5, 8, 11, or 15 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14,or 16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number PTA-1845, such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced into SEQ ID NO:1, 3, 4, 6,7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1845 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a DHDR protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a DHDR coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened forDHDR biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16,or the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number PTA-1845, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

In a preferred embodiment, a mutant DHDR protein can be assayed for theability to metabolize or catabolize biochemical molecules necessary forenergy production or storage, permit intra- or inter-cellular signaling,metabolize or catabolize metabolically important biomolecules, and todetoxify potentially harmful compounds.

In addition to the nucleic acid molecules encoding DHDR proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire DHDR coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a DHDR.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human DHDR corresponds to SEQ ID NO:3, 6, 9 or 12,and the coding region of mouse DHDR corresponds to SEQ ID NO:16). Thecoding region may or may not include a stop codon. In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding DHDR. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding DHDR disclosed herein (e.g.,SEQ ID NO:3, 6, 9, 12, or 16), antisense nucleic acids of the inventioncan be designed according to the rules of Watson and Crick base pairing.The antisense nucleic acid molecule can be complementary to the entirecoding region of DHDR mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof DHDR mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofDHDR mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a DHDR proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave DHDRmRNA transcripts to thereby inhibit translation of DHDR mRNA. A ribozymehaving specificity for a DHDR-encoding nucleic acid can be designedbased upon the nucleotide sequence of a DHDR cDNA disclosed herein(i.e., SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number PTA-1845). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina DHDR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, DHDR mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.(1993) Science 261:1411-1418.

Alternatively, DHDR gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the DHDR(e.g., the DHDR promoter and/or enhancers; e.g., nucleotides 1-62 of SEQID NO:1, nucleotides 1-330 of SEQ ID NO:4, nucleotides 1-280 of SEQ IDNO:7, nucleotides 1-60 of SEQ ID NO:10, or nucleotides 1-101 of SEQ IDNO:14) to form triple helical structures that prevent transcription ofthe DHDR gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

In yet another embodiment, the DHDR nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of DHDR nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of DHDR nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., Hyrup and Nielsen (1996) supra));or as probes or primers for DNA sequencing or hybridization (Hyrup andNielsen (1996) supra; Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of DHDR can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of DHDR nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup and Nielsen (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17):3357-63. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett.5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Biotechniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

Alternatively, the expression characteristics of an endogenous DHDR genewithin a cell line or microorganism may be modified by inserting aheterologous DNA regulatory element into the genome of a stable cellline or cloned microorganism such that the inserted regulatory elementis operatively linked with the endogenous DHDR gene. For example, anendogenous DHDR gene which is normally “transcriptionally silent”, i.e.,a DHDR gene which is normally not expressed, or is expressed only atvery low levels in a cell line or microorganism, may be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell line ormicroorganism. Alternatively, a transcriptionally silent, endogenousDHDR gene may be activated by insertion of a promiscuous regulatoryelement that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous DHDR gene, using techniques, such as targeted homologousrecombination, which are well known to those of skill in the art, anddescribed, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publicationNo. WO 91/06667, published May 16, 1991.

II. Isolated DHDR Proteins and Anti-DHDR Antibodies

One aspect of the invention pertains to isolated DHDR proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-DHDR antibodies. In oneembodiment, native DHDR proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, DHDR proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a DHDR protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theDHDR protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of DHDRprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of DHDR protein having less than about 30% (by dryweight) of non-DHDR protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-DHDR protein,still more preferably less than about 10% of non-DHDR protein, and mostpreferably less than about 5% non-DHDR protein. When the DHDR protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of DHDR protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of DHDR protein having less than about 30% (by dry weight)of chemical precursors or non-DHDR chemicals, more preferably less thanabout 20% chemical precursors or non-DHDR chemicals, still morepreferably less than about 10% chemical precursors or non-DHDRchemicals, and most preferably less than about 5% chemical precursors ornon-DHDR chemicals.

As used herein, a “biologically active portion” of a DHDR proteinincludes a fragment of a DHDR protein which participates in aninteraction between a DHDR molecule and a non-DHDR molecule.Biologically active portions of a DHDR protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the DHDR protein, e.g., the amino acidsequence shown in SEQ ID NO:2, 5, 8, 11, or 15, which include less aminoacids than the full length DHDR proteins, and exhibit at least oneactivity of a DHDR protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the DHDRprotein, e.g., modulating membrane excitability. A biologically activeportion of a DHDR protein can be a polypeptide which is, for example,25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800 or more amino acids in length. Biologicallyactive portions of a DHDR protein can be used as targets for developingagents which modulate a DHDR mediated activity, e.g., a proliferativeresponse.

In one embodiment, a biologically active portion of a DHDR proteincomprises at least one transmembrane domain. It is to be understood thata preferred biologically active portion of a DHDR protein of the presentinvention may contain at least one transmembrane domain and one or moreof the following domains: a signal peptide domain, an aldehydedehydrogenase oxidoreductase domain, an aldehyde dehydrogenase familydomain, a short chain dehydrogenase domain, an oxidoreductase proteindehydrogenase domain, a 3-beta hydroxysteroid dehydrogenase domain, aNAD-dependent epimerase/dehydratase domain, a short chaindehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain, adehydrogenase domain, or a glucose-1-dehydrogenase domain. Moreover,other biologically active portions, in which other regions of theprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native DHDRprotein.

In a preferred embodiment, the DHDR protein has an amino acid sequenceshown in SEQ ID NO:2, 5, 8, 11, or 15. In other embodiments, the DHDRprotein is substantially identical to SEQ ID NO:2, 5, 8, 11, or 15, andretains the functional activity of the protein of SEQ ID NO:2, 5, 8, 11,or 15, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described in detail in subsection I above.Accordingly, in another embodiment, the DHDR protein is a protein whichcomprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or more identical toSEQ ID NO:2, 5, 8, 11, or 15.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the DHDR amino acidsequence of SEQ ID NO:2, 5, 8, 11, or 15 having, e.g., 400 amino acidresidues, at least 120, preferably at least 160, more preferably atleast 200, even more preferably at least 240, and even more preferablyat least 270, 320, 360 or more amino acid residues are aligned). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available onlinethrough the website of the Genetics Computer Group), using either aBlosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available online through the website of the Genetics ComputerGroup), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70,or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment,the percent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0 or 2.0U), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to DHDR nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=100,wordlength=3 to obtain amino acid sequences homologous to DHDR proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the website of the NationalCenter for Biotechnology Information.

The invention also provides DHDR chimeric or fusion proteins. As usedherein, a DHDR “chimeric protein” or “fusion protein” comprises a DHDRpolypeptide operatively linked to a non-DHDR polypeptide. An “DHDRpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a DHDR molecule, whereas a “non-DHDR polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the DHDR protein, e.g.,a protein which is different from the DHDR protein and which is derivedfrom the same or a different organism. Within a DHDR fusion protein theDHDR polypeptide can correspond to all or a portion of a DHDR protein.In a preferred embodiment, a DHDR fusion protein comprises at least onebiologically active portion of a DHDR protein. In another preferredembodiment, a DHDR fusion protein comprises at least two biologicallyactive portions of a DHDR protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the DHDR polypeptideand the non-DHDR polypeptide are fused in-frame to each other. Thenon-DHDR polypeptide can be fused to the N-terminus or C-terminus of theDHDR polypeptide.

For example, in one embodiment, the fusion protein is a GST-DHDR fusionprotein in which the DHDR sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant DHDR.

In another embodiment, the fusion protein is a DHDR protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of DHDR can beincreased through use of a heterologous signal sequence.

The DHDR fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. TheDHDR fusion proteins can be used to affect the bioavailability of a DHDRsubstrate. Use of DHDR fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a DHDR protein; (ii)mis-regulation of the DHDR gene; and (iii) aberrant post-translationalmodification of a DHDR protein.

Moreover, the DHDR-fusion proteins of the invention can be used asimmunogens to produce anti-DHDR antibodies in a subject, to purify DHDRligands and in screening assays to identify molecules which inhibit theinteraction of DHDR with a DHDR substrate.

Preferably, a DHDR chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini, fillingin of cohesive ends as appropriate, alkaline phosphatase treatment toavoid undesirable joining, and enzymatic ligation. In anotherembodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). ADHDR-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the DHDR protein.

The present invention also pertains to variants of the DHDR proteinswhich function as either DHDR agonists (mimetics) or as DHDRantagonists. Variants of the DHDR proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a DHDRprotein. An agonist of the DHDR proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a DHDR protein. An antagonist of a DHDR protein caninhibit one or more of the activities of the naturally occurring form ofthe DHDR protein by, for example, competitively modulating aDHDR-mediated activity of a DHDR protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the DHDR protein.

In one embodiment, variants of a DHDR protein which function as eitherDHDR agonists (mimetics) or as DHDR antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a DHDR protein for DHDR protein agonist or antagonist activity. Inone embodiment, a variegated library of DHDR variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of DHDR variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential DHDR sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of DHDR sequences therein. There are avariety of methods which can be used to produce libraries of potentialDHDR variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential DHDR sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidsRes. 11:477.

In addition, libraries of fragments of a DHDR protein coding sequencecan be used to generate a variegated population of DHDR fragments forscreening and subsequent selection of variants of a DHDR protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a DHDR coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the DHDR protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of DHDR proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify DHDR variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delagrave et al. (1993) Protein Eng. 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated DHDR library. For example, a library of expression vectorscan be transfected into a cell line, e.g., a neuronal cell line, whichordinarily responds to a DHDR ligand in a particular DHDRligand-dependent manner. The transfected cells are then contacted with aDHDR ligand and the effect of expression of the mutant on, e.g.,membrane excitability of DHDR can be detected. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of signaling by the DHDR ligand, and the individual clonesfurther characterized.

An isolated DHDR protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind DHDR using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length DHDR protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of DHDR for use as immunogens. Theantigenic peptide of DHDR comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, or 15 andencompasses an epitope of DHDR such that an antibody raised against thepeptide forms a specific immune complex with the DHDR protein.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofDHDR that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity (see, for example,FIGS. 2, 7, 12, and 18).

A DHDR immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed DHDR protein or a chemicallysynthesized DHDR polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic DHDR preparation induces a polyclonal anti-DHDR antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-DHDRantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as a DHDR.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind DHDR molecules. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of DHDR. A monoclonal antibody composition thustypically displays a single binding affinity for a particular DHDRprotein with which it immunoreacts.

Polyclonal anti-DHDR antibodies can be prepared as described above byimmunizing a suitable subject with a DHDR immunogen. The anti-DHDRantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized DHDR. If desired, the antibody moleculesdirected against DHDR can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-DHDR antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med. 54:387-402; M. L. Gefter et al. (1977) SomaticCell Genet. 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a DHDR immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds DHDR.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-DHDR monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindDHDR, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-DHDR antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with DHDR to thereby isolateimmunoglobulin library members that bind DHDR. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hay et al. (1992)Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins etal. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580;Garrard et al. (1991) Biotechnology (NY) 9:1373-1377; Hoogenboom et al.(1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl.Acad. Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature348:552-554.

Additionally, recombinant anti-DHDR antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-DHDR antibody (e.g., monoclonal antibody) can be used to isolateDHDR by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-DHDR antibody can facilitate thepurification of natural DHDR from cells and of recombinantly producedDHDR expressed in host cells. Moreover, an anti-DHDR antibody can beused to detect DHDR protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the DHDR protein. Anti-DHDR antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a DHDR protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., DHDR proteins,mutant forms of DHDR proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of DHDR proteins in prokaryotic or eukaryotic cells. Forexample, DHDR proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel(1990) supra. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in DHDR activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for DHDR proteins, for example. In apreferred embodiment, a DHDR fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the DHDR expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, DHDR proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to DHDR mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which a DHDRnucleic acid molecule of the invention is introduced, e.g., a DHDRnucleic acid molecule within a recombinant expression vector or a DHDRnucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aDHDR protein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a DHDR protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a DHDR protein.Accordingly, the invention further provides methods for producing a DHDRprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of the invention (into which arecombinant expression vector encoding a DHDR protein has beenintroduced) in a suitable medium such that a DHDR protein is produced.In another embodiment, the method further comprises isolating a DHDRprotein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichDHDR-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous DHDRsequences have been introduced into their genome or homologousrecombinant animals in which endogenous DHDR sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a DHDR and for identifying and/or evaluating modulators ofDHDR activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous DHDR gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aDHDR-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The DHDRcDNA sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, orSEQ ID NO:14 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of a human DHDRgene, such as a mouse or rat DHDR gene, can be used as a transgene.Alternatively, a DHDR gene homologue, such as another DHDR familymember, can be isolated based on hybridization to the DHDR cDNAsequences of SEQ ID NO:1 or 3, SEQ ID NO:4 or 6, SEQ ID NO:7 or 9, SEQID NO:10 or 12, or SEQ ID NO:14 or 16, or the DNA insert of the plasmiddeposited with ATCC as Accession Number PTA-1845 (described further insubsection I above) and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to a DHDRtransgene to direct expression of a DHDR protein to particular cells.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a DHDR transgene in its genome and/or expression of DHDRmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a DHDRprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a DHDR gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the DHDR gene. The DHDR gene can be a human gene(e.g., the cDNA of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ IDNO:12), but more preferably, is a non-human homologue of a human DHDRgene (e.g., SEQ ID NO:16, or a cDNA isolated by stringent hybridizationwith the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 orSEQ ID NO:10). For example, a mouse DHDR gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous DHDR gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous DHDRgene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous DHDR gene is mutatedor otherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous DHDR protein). In the homologousrecombination nucleic acid molecule, the altered portion of the DHDRgene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the DHDR gene to allow for homologous recombination to occurbetween the exogenous DHDR gene carried by the homologous recombinationnucleic acid molecule and an endogenous DHDR gene in a cell, e.g., anembryonic stem cell. The additional flanking DHDR nucleic acid sequenceis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the homologous recombination nucleicacid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thehomologous recombination nucleic acid molecule is introduced into acell, e.g., an embryonic stem cell line (e.g., by electroporation) andcells in which the introduced DHDR gene has homologously recombined withthe endogenous DHDR gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells can then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Curr. Opin. Biotechnol. 2:823-829 and in PCTInternational Publication Nos. WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The DHDR nucleic acid molecules, fragments of DHDR proteins, andanti-DHDR antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of a DHDR protein or an anti-DHDR antibody)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating-plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention:

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It is

furthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

In certain embodiments of the invention, a modulator of DHDR activity isadministered in combination with other agents (e.g., a small molecule),or in conjunction with another, complementary treatment regime. Forexample, in one embodiment, a modulator of DHDR activity is used totreat DHDR associated disorder. Accordingly, modulation of DHDR activitymay be used in conjunction with, for example, another agent used totreat the disorder (e.g., another agent used to treat a viral orcellular proliferation disorder).

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a DHDR protein of the invention has one or more of thefollowing activities: 1) it modulates metabolism or catabolism ofbiochemical molecules necessary for energy production or storage, 2) itmodulates intra- or inter-cellular signaling, 3) it modulates metabolismor catabolism of metabolically important biomolecules (e.g.,glucocorticoids), 4) it modulates detoxification of potentially harmfulcompounds, 5) it modulates viral infection (e.g., by modulating viralgene expression), 6) it acts as a transcriptional cofactor for viralgene activation, and/or 7) it modulates viral activity, 8) it modulatescellular proliferation.

The isolated nucleic acid molecules of the invention can be used, forexample, to express DHDR protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect DHDR mRNA(e.g., in a biological sample) or a genetic alteration in a DHDR gene,and to modulate DHDR activity, as described further below. The DHDRproteins can be used to treat disorders characterized by insufficient orexcessive production of a DHDR substrate or production of DHDRinhibitors. In addition, the DHDR proteins can be used to screen fornaturally occurring DHDR substrates, to screen for drugs or compoundswhich modulate DHDR activity, as well as to treat disorderscharacterized by insufficient or excessive production of DHDR protein orproduction of DHDR protein forms which have decreased, aberrant orunwanted activity compared to DHDR wild type protein (e.g.,dehydrogenase-associated disorders, such as CNS disorders; cardiacdisorders; muscular disorders; cellular growth, differentiation, ormigration disorders; neurological disorders; immune disorders; hormonaldisorders; and viral disorders. Moreover, the anti-DHDR antibodies ofthe invention can be used to detect and isolate DHDR proteins, regulatethe bioavailability of DHDR proteins, and modulate DHDR activity.

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, e.g., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to DHDR proteins, have a stimulatory or inhibitory effect on,for example, DHDR expression or DHDR activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of DHDRsubstrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a DHDR protein or polypeptideor biologically active portion thereof (e.g., aldehydes, alcohols, orsteroids (e.g., glucocorticoids)). In another embodiment, the inventionprovides assays for screening candidate or test compounds which bind toor modulate the activity of a DHDR protein or polypeptide orbiologically active portion thereof (e.g., cofactor or coenzyme analogs,or inhibitory molecules). The test compounds of the present inventioncan be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a DHDR protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate DHDR activity is determined. Determining the ability of thetest compound to modulate DHDR activity can be accomplished bymonitoring, for example, the production of one or more specificmetabolites in a cell which expresses DHDR (see, e.g., Saada et al.(2000) Biochem. Biophys. Res. Commun. 269: 382-386). The cell, forexample, can be of mammalian origin, e.g., a liver cell, a neuronalcell, or a thymus cell.

The ability of the test compound to modulate DHDR binding to a substrate(e.g., an alcohol, an aldehyde, or a steroid (e.g., a glucocorticoid))or to bind to DHDR can also be determined. Determining the ability ofthe test compound to modulate DHDR binding to a substrate can beaccomplished, for example, by coupling the DHDR substrate with aradioisotope or enzymatic label such that binding of the DHDR substrateto DHDR can be determined by detecting the labeled DHDR substrate in acomplex. Alternatively, DHDR could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateDHDR binding to a DHDR substrate in a complex. Determining the abilityof the test compound to bind DHDR can be accomplished, for example, bycoupling the compound with a radioisotope or enzymatic label such thatbinding of the compound to DHDR can be determined by detecting thelabeled DHDR compound in a complex. For example, compounds (e.g., DHDRsubstrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directlyor indirectly, and the radioisotope detected by direct counting ofradioemission or by scintillation counting. Alternatively, compounds canbe enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a DHDR substrate) to interact with DHDR without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a compound with DHDR without thelabeling of either the compound or the DHDR. McConnell, H. M. et al.(1992) Science 257:1906-1912. As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and DHDR.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a DHDR target molecule (e.g., a DHDRsubstrate) with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of theDHDR target molecule. Determining the ability of the test compound tomodulate the activity of a DHDR target molecule can be accomplished, forexample, by determining the ability of the DHDR protein to bind to orinteract with the DHDR target molecule.

Determining the ability of the DHDR protein, or a biologically activefragment thereof, to bind to or interact with a DHDR target molecule canbe accomplished by one of the methods described above for determiningdirect binding. In a preferred embodiment, determining the ability ofthe DHDR protein to bind to or interact with a DHDR target molecule canbe accomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular response (i.e., changes inintracellular K⁺ levels or induction of viral gene expression),detecting catalytic/enzymatic activity of the target on an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a DHDR protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the DHDR protein or biologically active portionthereof is determined. Preferred biologically active portions of theDHDR proteins to be used in assays of the present invention includefragments which participate in interactions with non-DHDR molecules,e.g., fragments with high surface probability scores (see, for example,FIGS. 2, 7, 12, and 18). Binding of the test compound to the DHDRprotein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the DHDRprotein or biologically active portion thereof with a known compoundwhich binds DHDR to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a DHDR protein, wherein determining the ability of thetest compound to interact with a DHDR protein comprises determining theability of the test compound to preferentially bind to DHDR orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a DHDRprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the DHDR protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a DHDR protein can beaccomplished, for example, by determining the ability of the DHDRprotein to bind to a DHDR target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the DHDR protein to bind to a DHDR target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a DHDR protein can be accomplishedby determining the ability of the DHDR protein to further modulate theactivity of a downstream effector of a DHDR target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting aDHDR protein or biologically active portion thereof with a knowncompound which binds the DHDR protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the DHDR protein, whereindetermining the ability of the test compound to interact with the DHDRprotein comprises determining the ability of the DHDR protein topreferentially bind to or catalyze the transfer of a hydride moiety toor from the target substrate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either DHDR or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a DHDR protein, or interaction of aDHDR protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/DHDR fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or DHDR protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of DHDRbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a DHDRprotein or a DHDR target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated DHDR protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with DHDR protein or target molecules but which donot interfere with binding of the DHDR protein to its target moleculecan be derivatized to the wells of the plate, and unbound target or DHDRprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the DHDR protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the DHDR protein or target molecule.

In another embodiment, modulators of DHDR expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of DHDR mRNA or protein in the cell is determined. The levelof expression of DHDR mRNA or protein in the presence of the candidatecompound is compared to the level of expression of DHDR mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of DHDR expression based on thiscomparison. For example, when expression of DHDR mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of DHDR mRNA or protein expression.Alternatively, when expression of DHDR mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of DHDR mRNA or protein expression. The level of DHDR mRNA orprotein expression in the cells can be determined by methods describedherein for detecting DHDR mRNA or protein.

In yet another aspect of the invention, the DHDR proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with DHDR (“DHDR-binding proteins” or “DHDR-6-bp”) and areinvolved in DHDR activity. Such DHDR-binding proteins are also likely tobe involved in the propagation of signals by the DHDR proteins or DHDRtargets as, for example, downstream elements of a DHDR-mediatedsignaling pathway. Alternatively, such DHDR-binding proteins are likelyto be DHDR inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a DHDR protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a DHDR-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the DHDRprotein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a DHDR protein can be confirmedin vivo, e.g., in an animal such as an animal model for cellulartransformation and/or tumorigenesis or an animal model for viralinfection.

There are many animal models for viral infection known in the art. Forexample, a transgenic mouse model for hepatitis B virus infection (HBV)(Guidotti, L. G. et al. (1995) J. Virol. 69:6158-6169) may be used.High-level viral gene expression is present in the liver and kidneytissues of these mice, and the hepatocytes of the mice replicate thevirus at levels comparable to those in the infected livers of patientswith chronic hepatitis.

Another mouse model for HBV infection that may be used includes themouse model made by transplanting primary human hepatocytes into mice ina matrix under the kidney capsule along with administration of anagonistic antibody against c-Met (Ohashi, K. et al. (2000) Nat. Med.6:327-331). These mice are susceptible to HBV infection. Additionally,they are susceptible to super-infection with hepatitis delta virus(HDV).

Other mouse models for HBV infection that may be used include the micedescribed in Babinet, C. et al. (1985) Science 230:1160-3; Lee, T.-H. etal. (1990) J. Virol. 64:5939-5947; Madden, C. R. et al. (2000) J. Virol.74:5266-5272; Brown, J. J. et al. (2000) Hepatology 31:173-181; Larkin,J. (1999) Nat. Med. 5:907-912; and Araki, K. et al. (1989) Proc. Natl.Acad. Sci. USA 86:207-11.

Chronic HBV infection is a major risk factor for hepatocellularcarcinoma (Beasley, R. P. (1988) Cancer 61:1942-1956; Slagle, B. et al.(1994) In Viruses and cancer, Minson, A. et al., eds., University ofCambridge, Cambridge, England 51:149-171), and mice transgenic for theHBV X gene have increased sensitivity to hepatocarcinogens (Slagle, B.L. et al. (1996) Mol. Carcinog. 15:261-269). The double transgenic mousestrain described in Madden et al. (supra) can be used to study theeffects of test compounds identified by the screening methods of theinvention in modulating HBV X-mediated hepatocarcinogen sensitivity. Forexample, the mice can be treated with a hepatocarcinogen and a testcompound, and the effect of the test compound on thehepatocarcinogen-mediated mutation rate of the host DNA can be assayedby functional analysis of a bacteriophage lambda transgene. Briefly, DNAisolated from the livers of such treated mice can be packaged intolambda phage particles and used to infect E. coli bacteria. Mutationrates of the lambda particles (methods for determination of which areknown in the art) are directly related to the HBV X-mediated host DNAmutation rates in response to the hepatorcarcinogen in the treated mice.

Other mouse models for HBV infection and HBV immunity that may be usedinclude those made by transplanting human peripheral blood mononuclearcells (PBMC) from chronic HBV carriers and HBV-immunized donors,respectively, into lethally-irradiated Balb/c mice (Böcher, W. O. et al.(2000) Hepatology 31:480-487; Ilhan, E. et al. (1999) Hepatology29:553-562). Such human/mouse radiation chimeras, called Trimera mice,may be used to study the effects of test compounds identified by thescreening methods of the invention on human antibody and T cellresponses to HBV infection in vivo (Marcus, H. et al. (1995) Blood86:398-406; Reisner, Y. et al. (1998) Trends Biotechnol. 16:242-246;Segall, H. et al. (1996) Blood 88:721-730; Böcher, W. O. et al. (1999)Immunology 96:634-641).

The effects of a modulating agent on HBV infection can also be studiedin other hepadnavirus animal models: the woodchuck hepatitis virus (WHV)model (Korba, B. E. et al. (2000) Hepatology 31:1165-1175; Cote, P. J.et al. (2000) Hepatology 31:190-200), the duck hepatitis B virus (DHBV)model (Le Guerhier, F. et al. (2000) Antimicrob. Agents Chemother.44:111-122; Vickery, K. et al. (1999) J. Med. Virol. 58:19-25), and thechimpanzee and ground and tree squirrel models (Caselmann, W. H. (1994)Antiviral Res. 24:121-129).

While an animal model for hepatitis C virus (HCV) infection thatadequately reproduces the characteristics of HCV infection in humansdoes not yet exist, there is an HCV Trimera mouse model (Dekel, B. etal. (1995) J. Infect. Dis. 172:25-30), and there are some mouse strainsthat are transgenic for certain HCV proteins, and thus, may be usefulfor testing compounds that can modulate DHDR activity in vivo(Pasquinelli, C. et al. (1997) Hepatology 25:719-727).

Other animal models for viral infection are also known in the art andmay be used in the screening assays of the present invention. Forexample, there are many animal models for Epstein-Barr virus (EBV)associated lymphoproliferative disease. Such models have been made inrabbits, common marmosets (Callithrix jacchus), cottontop tamarins(Saguinus oedipus oedipus), rhesus monkeys, and the severe combinedimmunodeficient (SCID) mouse (Johannessen, I. and Crawford, D. H. (1999)Rev. Med. Virol. 9:263-77; Hayashi, K. and Akagi, T. (2000) Path.Intenational 50:85-97). The mouse γ-herpesvirus 68 infection model(Speck, S. H. and Virgin, H. W. (1999) Curr. Opin. Microbiol. 2:403-9;Virgin, H. W. and Speck, S. H. (1999) Curr. Opin. Immunol. 11:371-379)and the cotton rat model for measles virus infection (Niewiesk, S.(1999) Immunol. Lett. 65:47-50) present other examples of animal modelsthat may be used in the methods of the invention. Macaques infected withlive attenuated simian immunodeficiency virus (SIV) (Geretti, A. M.(1999) Rev. Med. Virol. 9:57-67; Almond, N. and Stott, J. (1999)Immunol. Lett. 66:167-170) as well as the chimpanzee HIV model (Murthy,K. K. et al. (1998) AIDS Res. Hum. Retroviruses 14 Suppl 3:S271-6) canbe used as models for human immunodeficiency virus (HIV) infection.

Other examples of animal models that may be used in the methods of theinvention include the transgenic mouse model for an AIDS-like disease(Renkema, H. G. and Saksela, K. (2000) Front. Biosci. 5:D268-83); thechicken model for lymphoma-inducing herpesviruses (Schat, K. A. andXing, Z. (2000) Dev. Comp. Immunol. 24:201-21); and the mouse model ofcytomegalovirus infection (Sweet, C. (1999) FEMS Microbiol. Rev.23:457-82).

In another embodiment of the invention, the ability of the agent tomodulate the activity of a DHDR protein can be tested in an animal suchas an animal model for a cellular proliferation disorder, e.g.,tumorigenesis. Animal based models for studying tumorigenesis in vivoare well known in the art (reviewed in Animal Models of CancerPredisposition Syndromes, Hiai, H. and Hino, 0. (eds.) 1999, Progress inExperimental Tumor Research, Vol. 35; Clarke, A. R. (2000)Carcinogenesis 21:435-41) and include, for example, carcinogen-inducedtumors (Rithidech, K. et al. (1999) Mutat. Res. 428:33-39; Miller, M. L.et al. (2000) Environ. Mol. Mutagen. 35:319-327), injection and/ortransplantation of tumor cells into an animal, as well as animalsbearing mutations in growth regulatory genes, for example, oncogenes(e.g., ras) (Arbeit, J. M. et al. (1993) Am. J. Pathol. 142:1187-1197;Sinn, E. et al. (1987) Cell 49:465-475; Thorgeirsson, S S et al. ToxicolLett (2000) 112-113:553-555) and tumor suppressor genes (e.g., p53)(Vooijs, M. et al. (1999) Oncogene 18:5293-5303; Clark A. R. (1995)Cancer Metast. Rev. 14:125-148; Kumar, T. R. et al. (1995) J. Intern.Med. 238:233-238; Donehower, L. A. et al. (1992) Nature 356215-221).Furthermore, experimental model systems are available for the study of,for example, ovarian cancer (Hamilton, T. C. et al. (1984) Semin. Oncol.11:285-298; Rahman, N. A. et al. (1998) Mol. Cell. Endocrinol.145:167-174; Beamer, W. G. et al. (1998) Toxicol. Pathol. 26:704-710),gastric cancer (Thompson, J. et al. (2000) Int. J. Cancer 86:863-869;Fodde, R. et al. (1999) Cytogenet. Cell Genet. 86:105-111), breastcancer (Li, M. et al. (2000) Oncogene 19:1010-1019; Green, J. E. et al.(2000) Oncogene 19:1020-1027), melanoma (Satyamoorthy, K. et al. (1999)Cancer Metast. Rev. 18:401-405), and prostate cancer (Shirai, T. et al.(2000) Mutat. Res. 462:219-226; Bostwick, D. G. et al. (2000) Prostate43:286-294).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model, as described above. For example, an agentidentified as described herein (e.g., a DHDR modulating agent, anantisense DHDR nucleic acid molecule, a DHDR-specific antibody, or aDHDR-binding partner) can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the DHDR nucleotide sequences, describedherein, can be used to map the location of the DHDR genes on achromosome. The mapping of the DHDR sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, DHDR genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the DHDR nucleotidesequences. Computer analysis of the DHDR sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the DHDR sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the DHDRnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a DHDRsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data (such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between a gene and adisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland, J. et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the DHDR gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The DHDR sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the DHDR nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The DHDR nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, SEQID NO:4, SEQ ID NO:7, or SEQ ID NO:10 can comfortably provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3, 6, 9, or 12are used, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from DHDR nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of DHDR Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7or SEQ ID NO:10 are particularly appropriate for this use as greaternumbers of polymorphisms occur in the noncoding regions, making iteasier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the DHDR nucleotide sequences orportions thereof, e.g., fragments derived from the noncoding regions ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10 having a length ofat least 20 bases, preferably at least 30 bases.

The DHDR nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., thymus or brain tissue. This can bevery useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such DHDR probes can be used toidentify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., DHDR primers or probes canbe used to screen tissue culture for contamination (i.e., screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining DHDR proteinand/or nucleic acid expression as well as DHDR activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant orunwanted DHDR expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with DHDR protein,nucleic acid expression or activity. For example, mutations in a DHDRgene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with DHDR protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of DHDR inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of DHDRprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting DHDR protein ornucleic acid (e.g., mRNA, or genomic DNA) that encodes DHDR protein suchthat the presence of DHDR protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting DHDR mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to DHDR mRNAor genomic DNA. The nucleic acid probe can be, for example, the DHDRnucleic acid set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16,or the DNA insert of the plasmid deposited with ATCC as Accession NumberPTA-1845, or a portion thereof, such as an oligonucleotide of at least15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to DHDR mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting DHDR protein is an antibody capable ofbinding to DHDR protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect DHDR mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of DHDR mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of DHDR proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of DHDR genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of DHDR protein includeintroducing into a subject a labeled anti-DHDR antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting DHDR protein, mRNA, orgenomic DNA, such that the presence of DHDR protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of DHDRprotein, mRNA or genomic DNA in the control sample with the presence ofDHDR protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of DHDRin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting DHDR protein or mRNA in abiological sample; means for determining the amount of DHDR in thesample; and means for comparing the amount of DHDR in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectDHDR protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted DHDR expression or activity. Asused herein, the term “aberrant” includes a DHDR expression or activitywhich deviates from the wild type DHDR expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant DHDR expression or activity isintended to include the cases in which a mutation in the DHDR genecauses the DHDR gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional DHDRprotein or a protein which does not function in a wild-type fashion,e.g., a protein which does not interact with a DHDR substrate, or onewhich interacts with a non-DHDR substrate. As used herein, the term“unwanted” includes an unwanted phenomenon involved in a biologicalresponse such as cellular proliferation. For example, the term unwantedincludes a DHDR expression or activity which is undesirable in asubject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in DHDRprotein activity or nucleic acid expression, such as a CNS disorder(e.g., a cognitive or neurodegenerative disorder), a cellularproliferation, growth, differentiation, or migration disorder, acardiovascular disorder, musculoskeletal disorder, an immune disorder, aviral disorder, or a hormonal disorder. Alternatively, the prognosticassays can be utilized to identify a subject having or at risk fordeveloping a disorder associated with a misregulation in DHDR proteinactivity or nucleic acid expression, such as a CNS disorder, a cellularproliferation, growth, differentiation, or migration disorder, amusculoskeletal disorder, a cardiovascular disorder, an immune disorder,a viral disorder, or a hormonal disorder. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant or unwanted DHDR expression or activity in which a test sampleis obtained from a subject and DHDR protein or nucleic acid (e.g., mRNAor genomic DNA) is detected, wherein the presence of DHDR protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant or unwanted DHDRexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., cerebrospinal fluid orserum), cell sample, or tissue sample (e.g., a liver sample).

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted DHDR expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a CNS disorder, a musculardisorder, a cellular proliferation, growth, differentiation, ormigration disorder, an immune disorder, a viral disorder, or a hormonaldisorder. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant or unwanted DHDR expression oractivity in which a test sample is obtained and DHDR protein or nucleicacid expression or activity is detected (e.g., wherein the abundance ofDHDR protein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted DHDR expression or activity).

The methods of the invention can also be used to detect geneticalterations in a DHDR gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inDHDR protein activity or nucleic acid expression, such as a CNSdisorder, a musculoskeletal disorder, a cellular proliferation, growth,differentiation, or migration disorder, a cardiovascular disorder, animmune disorder, a viral disorder, or a hormonal disorder. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a DHDR-protein, or the mis-expression of the DHDRgene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a DHDR gene; 2) an addition of one or morenucleotides to a DHDR gene; 3) a substitution of one or more nucleotidesof a DHDR gene, 4) a chromosomal rearrangement of a DHDR gene; 5) analteration in the level of a messenger RNA transcript of a DHDR gene, 6)aberrant modification of a DHDR gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a DHDR gene, 8) a non-wild type levelof a DHDR-protein, 9) allelic loss of a DHDR gene, and 10) inappropriatepost-translational modification of a DHDR-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a DHDR gene. A preferred biological sampleis a tissue (e.g., a liver sample) or serum sample isolated byconventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a DHDR gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a DHDR gene under conditions such thathybridization and amplification of the DHDR gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a DHDR gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in DHDR can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J.et al. (1996) Nat. Med. 2:753-759). For example, genetic mutations inDHDR can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. (1996) supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the DHDR gene anddetect mutations by comparing the sequence of the sample DHDR with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the DHDR gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type DHDR sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in DHDR cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a DHDR sequence,e.g., a wild-type DHDR sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in DHDR genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA:86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control DHDRnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a DHDR gene.

Furthermore, any cell type or tissue in which DHDR is expressed may beutilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a DHDR protein (e.g., the modulation of a virus activityand/or the modulation of cell proliferation and/or migration) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase DHDR gene expression, proteinlevels, or upregulate DHDR activity, can be monitored in clinical trialsof subjects exhibiting decreased DHDR gene expression, protein levels,or downregulated DHDR activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease DHDR gene expression,protein levels, or downregulate DHDR activity, can be monitored inclinical trials of subjects exhibiting increased DHDR gene expression,protein levels, or upregulated DHDR activity. In such clinical trials,the expression or activity of a DHDR gene, and preferably, other genesthat have been implicated in, for example, a DHDR-associated disordercan be used as a “read out” or markers of the phenotype of a particularcell.

For example, and not by way of limitation, genes, including DHDR, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates DHDR activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on DHDR-associated disorders (e.g., disorderscharacterized by viral infection and/or deregulated cell proliferationand/or migration), for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofDHDR and other genes implicated in the DHDR-associated disorder,respectively. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of DHDR or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug-candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a DHDR protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the DHDR protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the DHDR protein, mRNA, or genomic DNA inthe pre-administration sample with the DHDR protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of DHDR to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of DHDR to lower levels than detected, i.e., to decrease theeffectiveness of the agent. According to such an embodiment, DHDRexpression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

D. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted DHDRexpression or activity, e.g., a dehydrogenase-associated disorder suchas a CNS disorder; a cellular proliferation, growth, differentiation, ormigration disorder; a, musculoskeletal disorder; a cardiovasculardisorder; an immune disorder; a viral disorder; or a hormonal disorder.As used herein, “treatment” of a subject includes the application oradministration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to a cell or tissue from asubject, who has a diseases or disorder, has a symptom of a disease ordisorder, or is at risk of (or susceptible to) a disease or disorder,with the purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disease or disorder, the symptom ofthe disease or disorder, or the risk of (or susceptibility to) thedisease or disorder. As used herein, a “therapeutic agent” includes, butis not limited to, small molecules, peptides, polypeptides, antibodies,ribozymes, and antisense oligonucleotides.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the DHDR molecules ofthe present invention or DHDR modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedDHDR expression or activity, by administering to the subject a DHDR oran agent which modulates DHDR expression or at least one DHDR activity.Subjects at risk for a disease which is caused or contributed to byaberrant or unwanted DHDR expression or activity can be identified by,for example, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the DHDR aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of DHDR aberrancy, forexample, a DHDR, DHDR agonist or DHDR antagonist agent can be used fortreating the subject. The appropriate agent can be determined based onscreening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating DHDRexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a DHDR or agent that modulates one or more of theactivities of DHDR protein activity associated with the cell. An agentthat modulates DHDR protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a DHDR protein (e.g., a DHDR substrate), a DHDRantibody, a DHDR agonist or antagonist, a peptidomimetic of a DHDRagonist or antagonist, or other small molecule. In one embodiment, theagent stimulates one or more DHDR activities. Examples of suchstimulatory agents include active DHDR protein and a nucleic acidmolecule encoding DHDR that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more DHDR activities.Examples of such inhibitory agents include antisense DHDR nucleic acidmolecules, anti-DHDR antibodies, and DHDR inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a DHDR protein or nucleicacid molecule. In one embodiment, the method involves administering anagent (e.g., an agent identified by a screening assay described herein),or combination of agents that modulates (e.g., upregulates ordownregulates) DHDR expression or activity. In another embodiment, themethod involves administering a DHDR protein or nucleic acid molecule astherapy to compensate for reduced, aberrant, or unwanted DHDR expressionor activity.

Stimulation of DHDR activity is desirable in situations in which DHDR isabnormally downregulated and/or in which increased DHDR activity islikely to have a beneficial effect. Likewise, inhibition of DHDRactivity is desirable in situations in which DHDR is abnormallyupregulated and/or in which decreased DHDR activity is likely to have abeneficial effect.

3. Pharmacogenomics

The DHDR molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on DHDRactivity (e.g., DHDR gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) DHDR-associated disorders (e.g.,proliferative disorders, CNS disorders, cardiac disorders, metabolicdisorders, or muscular disorders) associated with aberrant or unwantedDHDR activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer aDHDR molecule or DHDR modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a DHDR molecule or DHDR modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a DHDRprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a DHDR molecule orDHDR modulator of the present invention) can give an indication whethergene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a DHDR molecule orDHDR modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

E. Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising DHDR sequence informationis also provided. As used herein, “DHDR sequence information” refers toany nucleotide and/or amino acid sequence information particular to theDHDR molecules of the present invention, including but not limited tofull-length nucleotide and/or amino acid sequences, partial nucleotideand/or amino acid sequences, polymorphic sequences including singlenucleotide polymorphisms (SNPs), epitope sequences, and the like.Moreover, information “related to” said DHDR sequence informationincludes detection of the presence or absence of a sequence (e.g.,detection of expression of a sequence, fragment, polymorphism, etc.),determination of the level of a sequence (e.g., detection of a level ofexpression, for example, a quantitative detection), detection of areactivity to a sequence (e.g., detection of protein expression and/orlevels, for example, using a sequence-specific antibody), and the like.As used herein, “electronic apparatus readable media” refers to anysuitable medium for storing, holding, or containing data or informationthat can be read and accessed directly by an electronic apparatus. Suchmedia can include, but are not limited to: magnetic storage media, suchas floppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as compact discs; electronic storage media such asRAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybridsof these categories such as magnetic/optical storage media. The mediumis adapted or configured for having recorded thereon DHDR sequenceinformation of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatuses; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe DHDR sequence information.

A variety of software programs and formats can be used to store thesequence information on the electronic apparatus readable medium. Forexample, the sequence information can be represented in a wordprocessing text file, formatted in commercially-available software suchas WordPerfect and Microsoft Word, represented in the form of an ASCIIfile, or stored in a database application, such as DB2, Sybase, Oracle,or the like, as well as in other forms. Any number of dataprocessorstructuring formats (e.g., text file or database) may be employed inorder to obtain or create a medium having recorded thereon the DHDRsequence information.

By providing DHDR sequence information in readable form, one canroutinely access the sequence information for a variety of purposes. Forexample, one skilled in the art can use the sequence information inreadable form to compare a target sequence or target structural motifwith the sequence information stored within the data storage means.Search means are used to identify fragments or regions of the sequencesof the invention which match a particular target sequence or targetmotif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, wherein the method comprises the stepsof determining DHDR sequence information associated with the subject andbased on the DHDR sequence information, determining whether the subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, and/or recommending a particulartreatment for the disease, disorder, or pre-disease condition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has a DHDRassociated disease or disorder or a pre-disposition to a diseaseassociated with DHDR wherein the method comprises the steps ofdetermining DHDR sequence information associated with the subject, andbased on the DHDR sequence information, determining whether the subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, and/or recommending a particulartreatment for the disease, disorder or pre-disease condition. The methodmay further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

The present invention also provides in a network, a method fordetermining whether a subject has a DHDR associated disease or disorderor a pre-disposition to a DHDR associated disease or disorder associatedwith DHDR, said method comprising the steps of receiving DHDR sequenceinformation from the subject and/or information related thereto,receiving phenotypic information associated with the subject, acquiringinformation from the network corresponding to DHDR and/or a DHDRassociated disease or disorder, and based on one or more of thephenotypic information, the DHDR information (e.g., sequence informationand/or information related thereto), and the acquired information,determining whether the subject has a DHDR associated disease ordisorder or a pre-disposition to a DHDR associated disease or disorder.The method may further comprise the step of recommending a particulartreatment for the disease, disorder or pre-disease condition.

The present invention also provides a business method for determiningwhether a subject has a DHDR associated disease or disorder or apre-disposition to a DHDR associated disease or disorder, said methodcomprising the steps of receiving information related to DHDR (e.g.,sequence information and/or information related thereto), receivingphenotypic information associated with the subject, acquiringinformation from the network related to DHDR and/or related to a DHDRassociated disease or disorder, and based on one or more of thephenotypic information, the DHDR information, and the acquiredinformation, determining whether the subject has a DHDR associateddisease or disorder or a pre-disposition to a DHDR associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder or pre-disease condition.

The invention also includes an array comprising a DHDR sequence of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be DHDR. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative determination, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined. Such a determination is useful,for example, to know the effect of cell-cell interaction at the level ofgene expression. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a DHDR associated disease or disorder, progression ofDHDR associated disease or disorder, and processes, such a cellulartransformation associated with the DHDR associated disease or disorder.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., ascertaining the effect of DHDR expression on theexpression of other genes). This provides, for example, for a selectionof alternate molecular targets for therapeutic intervention if theultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including DHDR) that could serve as amolecular target for diagnosis or therapeutic intervention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures and the sequence listing, areincorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human andMouse DHDR cDNA

In this example, the identification and characterization of the genesencoding human DHDR-1 (clone Fbh32142), DHDR-2 (clone Fbh21481), DHDR-3(clone Fbh25964), and DHDR-4 (clone Fbh21686), and mouse DHDR-2 (clonem21481) is described.

Isolation of the DHDR cDNA

The invention is based, at least in part, on the discovery of severalhuman and mouse genes encoding novel proteins, referred to herein asDHDR-1, DHDR-2, DHDR-3 and DHDR-4. The entire sequences of human clonesFbh32142, Fbh21481, Fbh25964, and Fbh21686, and mouse clone m21481 weredetermined and found to contain open reading frames termed human“DHDR-1”, “DHDR-2”, “DHDR-3”, and “DHDR-4”, and mouse “DHDR-2”,respectively, set forth in FIGS. 1, 6, 11, 16, and 31A, respectively.The amino acid sequences of these human and mouse DHDR expressionproducts are set forth in FIGS. 1, 6, 11, 16, and 31B, respectively. Thehuman DHDR-1 protein sequence set forth in SEQ ID NO:2 comprises about802 amino acid residues and is shown in FIGS. 1A-1D. The human DHDR-2protein sequence set forth in SEQ ID NO:5 comprises about 311 amino acidresidues and is shown in FIGS. 6A-6B. The human DHDR-3 protein sequenceset forth in SEQ ID NO:8 comprises about 369 amino acid residues and isshown in Figures 11A-11B. The human DHDR-4 protein sequence set forth inSEQ ID NO:11 comprises about 322 amino acid residues and is shown inFIG. 16. The mouse DHDR-2 protein set forth in SEQ ID NO:15 comprisesabout 311 amino acid residues and is shown in FIG. 31B. The codingregions (open reading frames) of SEQ ID NOs:1, 4, 7, 10, and 14 are setforth as SEQ ID NOs:3, 6, 9, 12, and 16. Clones Fbh21481, and Fbh21686,comprising the coding regions of human DHDR-2, and DHDR-4, respectively,were deposited with the American Type Culture Collection (ATCC®), 10801University Boulevard, Manassas, Va. 20110-2209, on May 9, 2000, and Mar.22, 2001, respectively, and assigned Accession Nos. PTA-1845, and______, respectively.

Analysis of the Human and Mouse DHDR Molecules

The amino acid sequences of human DHDR-1, DHDR-2, DHDR-3, and DHDR-4were analyzed using the program PSORT (available online through thePSORT server website) to predict the localization of the proteins withinthe cell. This program assesses the presence of different targeting andlocalization amino acid sequences within the query sequence. The resultsof the analyses show that human DHDR-1 (SEQ ID NO:2) may be localized tothe mitochondrion, to the endoplasmic reticulum, to the nucleus, or tosecretory vesicles. The results of the analyses further show that humanDHDR-2 (SEQ ID NO:5) may be localized to the mitochondrion, to thecytoplasm, to extracellular spaces or the cell wall, to vacuoles, to thenucleus, or to the endoplasmic reticulum. The results of the analysesfurther show that human DHDR-3 (SEQ ID NO:8) may be localized to thecytoplasm, to the mitochondrion, to the Golgi, to the endoplasmicreticulum, to the extracellular space or cell wall, to vacuoles, to thenucleus, or to secretory vesicles. The results of the analyses furthershow that human DHDR-4 (SEQ ID NO:11) may be localized to the nucleus,the cytoplasm, to the Golgi, to the mitochondrion, to peroxisomes, tothe endoplasmic reticulum, or to secretory vesicles.

An alignment of the human DHDR-4 amino acid sequence with the amino acidsequence of Rattus norvegicus putative short-chaindehydrogenase/reductase (Accession Number AF099742) using the CLUSTAL W(1.74) multiple sequence alignment program is set forth in FIG. 17.

Each of the amino acid sequences of human DHDR-1, DHDR-2, DHDR-3, andDHDR-4 were analyzed by the SignalP program (Henrik et al. (1997) Prot.Eng. 10:1-6) for the presence of a signal peptide. These analysesrevealed the presence of a signal peptide in the amino acid sequence ofhuman DHDR-2 from residues 1-18 (FIG. 8). These analyses furtherrevealed the possible presence of a signal peptide in the amino acidsequence of human DHDR-4, from residues 1-19 (FIG. 19).

Searches of each of the amino acid sequences of human DHDR-1, DHDR-2,DHDR-3, and DHDR-4 were performed against the Memsat database (FIGS. 3,8, 13, and 19). These searches resulted in the identification of onetransmembrane domain in the amino acid sequence of human DHDR-1 (SEQ IDNO:2) at about residues 159-175, and one transmembrane domain in theamino acid sequence of human DHDR-2 (SEQ ID NO:5) at about residues 7-23in the native molecule, or about residues 265-283 of the predictedmature protein. These searches further identified four transmembranedomains in the amino acid sequence of human DHDR-3 (SEQ ID NO:8) atabout residues 10-26, 73-90, 289-305, and 312-333, and fourtransmembrane domains in the amino acid sequence of human DHDR-4 (SEQ IDNO:11) at about residues 29-50, 170-188, 208-224, and 258-275 of thenative molecule, and at about residues 10-31, 151-169, 189-205, and239-256 of the predicted mature protein.

Searches of each of the amino acid sequences of human DHDR-1, DHDR-2,DHDR-3, and DHDR-4 were also performed against the HMM database (FIGS.4, 9, 14, and 20). These searches resulted in the identification of an“aldehyde dehydrogenase family domain” in the amino acid sequence ofhuman DHDR-1 (SEQ ID NO:2) at about residues 47-494 (score=149.8) (FIG.4); the identification of a “short-chain dehydrogenase domain” in theamino acid sequence of human DHDR-2 (SEQ ID NO:5) at about residues38-227 (score=120.0) (FIG. 9), and the identification of a “3-betahydroxysteroid dehydrogenase domain” at about residues 1-365(score=676.9), a “short chain dehydrogenase domain” at about residues10-197, and a “NAD-dependent epimerase/dehydratase domain” at aboutresidues 12-365 of the amino acid sequence of human DHDR-3 (SEQ ID NO:8)(FIGS. 14A-14B). These searches further resulted in the identificationof a “short chain dehydrogenase domain” at about residues 38-226(score=162.5), and a “short chain dehydrogenase/reductase domain” atabout residues 250-280 (score=47.2) of the amino acid sequence of humanDHDR-4 (SEQ ID NO:11) (FIG. 20).

Searches of each of the amino acid sequences of human DHDR-1, DHDR-2,DHDR-3, and DHDR-4 were also performed against the ProDom database(FIGS. 5, 10, 15, and 21). These searches resulted in the identificationof an “aldehyde dehydrogenase oxidoreductase domain” in the amino acidsequence of human DHDR-1 (SEQ ID NO:2) at about residues 101-770(score=280) (FIGS. 5A-5B), and the identification of an “oxidoreductaseprotein dehydrogenase domain” in the amino acid sequence of human DHDR-2(SEQ ID NO:5) at about residues 99-219 (score=113) (FIG. 10). Thesesearches further resulted in the identification of a “3-betahydroxysteroid dehydrogenase domain” in the amino acid sequence of humanDHDR-3 (SEQ ID NO:8) at about residues 11-362 (score=395) (FIG. 15).These searches further resulted in the identification of an“oxidoreductase protein dehydrogenase domain” at about residues 37-231(score=157), a “shikimate 5-dehydrogenase domain” at about residues35-82 (score=86), a “dehydrogenase domain” at about residues 237-286(score=84), and a “glucose-1dehydrogenase domain” at about residues243-287 (score=92) of the amino acid sequence of human DHDR-4 (SEQ IDNO:11) (FIGS. 21A-21B).

The nucleotide sequences of the mouse and human DHDR-2 genes (SEQ IDNOs:14 and 4, respectively) were aligned using the GAP program in theGCG software package (nwsgapdna.cmp matrix) with a gap weight of 12 anda length weight of 4. The results are shown in FIGS. 32A-32B. As shownin the alignment, the mouse and human DHDR-2 nucleotide sequences areabout 88.1% identical.

The amino acid sequences of the mouse and human DHDR-2 genes (SEQ IDNOs:15 and 5, respectively) were aligned FIG. 33 depicts an alignment ofthe mouse DHDR-2 amino acid sequence using the GAP program in the GCGsoftware package (Blosum 62 matrix) with a gap weight of 12 and a lengthweight of 4. The results are shown in FIG. 33. As shown in thealignment, the mouse and human DHDR-2 amino acid sequences are about91.3% identical.

Analysis of DHDR mRNA Expression

This example describes the expression of human DHDR mRNA in varioustissues, cell lines, and disease models, as determined using the TaqMan™procedure and ill situ hybridization analysis.

The Taqman™ procedure is a quantitative, real-time PCR-based approach todetecting mRNA. The RT-PCR reaction exploits the 5′ nuclease activity ofAmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probe during PCR.Briefly, cDNA was generated from the samples of interest and served asthe starting material for PCR amplification. In addition to the 5′ and3′ gene-specific primers, a gene-specific oligonucleotide probe(complementary to the region being amplified) was included in thereaction (i.e., the Taqman™ probe). The TaqMan™ probe included anoligonucleotide with a fluorescent reporter dye covalently linked to the5′ end of the probe (such as FAM (6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separated the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products was detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe was intact, the proximity of the reporter dye to the quencher dyeresulted in suppression of the reporter fluorescence. During PCR, if thetarget of interest was present, the probe specifically annealed betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaved the probe between the reporterand the quencher only if the probe hybridized to the target. The probefragments were then displaced from the target, and polymerization of thestrand continued. The 3′ end of the probe was blocked to preventextension of the probe during PCR. This process occurred in every cycleand did not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlGAPDH or β-actin gene confirming efficient removal of genomic DNAcontamination.

For in situ analysis, various tissues, e.g., tissues obtained from liveror colon, were first frozen on dry ice. Ten-micrometer-thick sections ofthe tissues were postfixed with 4% formaldehyde in DEPC treated 1×phosphate-buffered saline (PBS) at room temperature for 10 minutesbefore being rinsed twice in DEPC 1× phosphate-buffered saline and oncein 0.1 M triethanolamine-HCl (pH 8.0). Following incubation in 0.25%acetic anhydride-0.1 M triethanolamine-HCl for 10 minutes, sections wererinsed in DEPC 2×SSC (1×SSC is 0.15M NaCl plus 0.015M sodium citrate).Tissues were then dehydrated through a series of ethanol washes,incubated in 100% chloroform for 5 minutes, and then rinsed in 100%ethanol for 1 minute and 95% ethanol for 1 minute and allowed to airdry.

Hybridizations were performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes were incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides were washed with 2×SSC. Sections were thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides were then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections were then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Human DHDR-1

The human DHDR-1 gene is highly expressed in coronary smooth musclecells (SMCs), human umbilical vein endothelial cells (HUVECs), kidney,skeletal muscle, adipose tissue, pancreas, skin, brain (cortex andhypothalamus), dorsal root ganglion (DRG), breast, ovary, prostate,prostate epithelial cells, colon, fibrotic liver, tonsil, and decubitusskin (see FIG. 22). The human DHDR-1 gene is expressed at lower levelsin a number of other tissues (see FIG. 22).

The human DHDR-1 gene is also expressed in a number of tumorigenic celllines, including MCF-7 (breast tumor), ZR75 (breast tumor), T47D (breasttumor), MDA 231 (breast tumor), MDA 435 (breast tumor), SKBr3 (breasttumor), DLD 1 (colon tumor—stage C), SW620 (colon tumor—stage C), HCT116(colon tumor), HT29 (colon tumor), Colo 205, NCIH125, NCIH322, NCIH460,A549, NHBE, SKOV-3 (ovary tumor), and OVCAR-3 (ovary tumor).

Expression of the human DHDR-1 gene is upregulated in 3/5 ovary tumors(FIG. 23), as compared to normal ovary. In situ hybridizationexperiments also showed an upregulation of human DHDR-2 in 1 borderlineovary tumor and 2/2 invasive ovary tumors.

Expression of the human DHDR-1 gene is upregulated in 6/7 lung tumors(FIG. 23), as compared to normal lung. In situ hybridization experimentsalso showed an upregulation in 1/2 lung tumors.

Expression of the human DHDR-1 gene is upregulated in 6/15hyperplastic/dysplastic lesions in the colon (FIG. 24), 3/4 colon tumors(FIG. 23), and 12/13 colon tumor metastases to the liver (FIGS. 23 and24), as compared to the expression in normal colon and liver. In situhybridization experiments also showed a marked elevation of expressionas early as hyperplasia/dysplasia (1/1 hyperplastic/dysplastic lesions),which is maintained in primary tumors (5/5 tumors) and metastases (3/3metastases to the liver).

Expression of the human DHDR-1 gene is upregulated in NOC synchronizedHCT116 cells (colon tumor derived cell line) at the time point t=24after entry into the cell cycle (FIG. 25). The time point t=0 signifiesthe G2/M border.

Human DHDR-2

Expression of the human DHDR-2 gene is downregulated in 7/7 breasttumors (FIG. 26), as compared to the expression in normal breast;downregulated in 6/6 colon tumors and 4/4 colon tumor metastases to theliver (FIG. 26), as compared to the expression in normal colon andliver; and downregulated in 3/3 glioblastoma brain tumors (FIG. 26).

Human DHDR-4

The human DHDR-4 gene is highly expressed in liver, kidney, brain, skin,prostate epithelial cells, primary osteoblasts, pituitary, CaCO cells,keratinocytes, aortic endothelial cells, fetal kidney, fetal lung,mammary epithelium, fetal spleen, fetal liver, umbilical smooth muscle,RAII Burkitt Lymphoma cells, lung, prostate, K53 red blood cells, fetaldorsal spinal cord, insulinoma cells, normal breast and ovarianepithelia, retina, HMC-1 mast cells, ovarian ascites, d8 dendriticcells, megakaryocytes, human mobilized bone morrow, mammary carcinoma,in melanoma cells, lymph, vein, U937/A70p B cells, A549con cells, WT LNCap testosterone cells, and esophagus. Significant expression of DHDR-4was also observed in aorta, breast, liver, lung, small intestine, glialcells, and thymus. Some expression of DHDR-4 was observed in brain,cervix, colon, heart, kidney, muscle, ovary, placenta, testes, andthyroid.

Human DHDR-4 is also greatly induced in situations of hepatitis B virus(HBV) infection (FIG. 28). Human DHDR-4 is expressed at 4-18 fold higherlevels in HBV-infected liver than in normal liver. The upregulation inHBV infected liver is specific to HBV; no upregulation of human DHDR-4is seen in hepatitis C virus (HCV) infected liver (FIG. 28). HumanDHDR-4 expression levels are 12-25 fold higher in HBV-expressingHepG2.2.15 cells than in HepG2 control cells (FIG. 28). Treatment ofHepG2 cells with Bayer compound IC50 or IC100 results in a stronginduction of human DHDR-4 expression, while treatment of HBV-expressingHepG2.2.15 cells with Bayer compound IC50 or IC100results in a strongdecrease in expression of human DHDR-4. Transfection of the HBV Xtranscription factor alone can induce a 5-fold increase in DHDR-4expression (FIG. 28). In situ hybridization analysis also revealed thathuman DHDR-4 is expressed at a much higher level in HBV positive liverthan in normal liver.

The human DHDR-4 gene is expressed in a number of tumorigenic celllines, including MCF-7 (breast tumor), ZR75 (breast tumor), T47D (breasttumor), MDA 231 (breast tumor), MDA 435 (breast tumor), DLD 1 (colontumor—stage C), SW 480, SW 620 (colon tumor—stage C), HCT 116 (colontumor), HT 29 (colon tumor), Colo 205, NCIH 125, NCIH 67, NCIH 322, andA549.

Expression of human DHDR-4 is downregulated in 6/6 glioblastoma braintumors (FIG. 29), as compared to normal brain. Expression of humanDHDR-4 is downregulated in 6/6 breast tumors, as compared to normalbreast (FIG. 30).

Expression of human DHDR-4 is upregulated in 6/8 lung tumors, ascompared to normal lung (FIG. 30). In situ data confirmed that humanDHDR-4 is upregulated in poorly differentiated non-small cell carcinomaof the lung (PD NSCCL).

Expression of human DHDR-4 is downregulated in synchronized A549 cellsat the time point t=6 after entering the cell cycle, and upregulated att=12 (FIG. 30). The time point t=0 signifies the G2/M border.

Example 2 Expression of Recombinant DHDR Protein in Bacterial Cells

In this example, DHDR is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, DHDR isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-DHDR fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 3 Expression of Recombinant DHDR Protein in COS Cells

To express the DHDR gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire DHDR protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the DHDR DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the DHDR codingsequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the DHDR coding sequence. The PCR amplified fragmentand the pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the DHDR gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the DHDR-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the DHDR polypeptide is detected by radiolabeling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine).The culture media are then collected and the cells are lysed usingdetergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50mM Tris, pH 7.5). Both the cell lysate and the culture media areprecipitated with an HA-specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the DHDR coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the DHDRpolypeptide is detected by radiolabeling and immunoprecipitation using aDHDR specific monoclonal antibody.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence which isat least 60% identical to the nucleotide sequence of SEQ ID NO:1 or SEQID NO:3, SEQ ID NO:7 or SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:12, orSEQ ID NO:14 or SEQ ID NO:16, or a complement thereof; b) a nucleic acidmolecule which encodes a polypeptide comprising an amino acid sequencewhich is at least 60% identical to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15; c) a nucleic acidmolecule which encodes a fragment of a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ IDNO:15, wherein the fragment comprises at least 16 contiguous amino acidsof SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15; and d) anucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15, wherein the nucleicacid molecule hybridizes to a nucleic acid molecule comprising SEQ IDNO:1 or SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:9, SEQ ID NO:10 or SEQ IDNO:12, or SEQ ID NO:14 or SEQ ID NO:16, or a complement thereof, understringent conditions.
 2. The isolated nucleic acid molecule of claim 1,further comprising a nucleotide sequence encoding a heterologouspolypeptide.
 3. A vector comprising the nucleic acid molecule ofclaim
 1. 4. A host cell comprising the vector of claim
 3. 5. A method ofproducing a polypeptide comprising culturing the host cell of claim 4 inan appropriate culture medium to thereby produce the polypeptide.
 6. Anisolated polypeptide selected from the group consisting of: a) afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15, wherein the fragmentcomprises at least 16 contiguous amino acids of SEQ ID NO:2, SEQ IDNO:8, SEQ ID NO:11, or SQ ID NO:15; b) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to acomplement of a nucleic acid molecule consisting of SEQ ID NO:1 or SEQID NO:3, SEQ ID NO:7 or SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:12, orSEQ ID NO:14 or SEQ ID NO:16 under stringent conditions; c) apolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 60% identical to a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ IDNO:16; d) a polypeptide comprising an amino acid sequence which is atleast 60% identical to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:8, SEQ ID NO:11, or SEQ ID NO:15.
 7. The isolated polypeptide ofclaim 6 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8,or SEQ ID NO:11.
 8. The polypeptide of claim 6, further comprisingheterologous amino acid sequences.
 9. An antibody which selectivelybinds to a polypeptide of claim
 6. 10. A method for detecting thepresence of a polypeptide of claim 6 in a sample comprising: a)contacting the sample with a compound which selectively binds to thepolypeptide; and b) determining whether the compound binds to thepolypeptide in the sample to thereby detect the presence of apolypeptide of claim 6 in the sample.
 11. The method of claim 10,wherein the compound which binds to the polypeptide is an antibody. 12.A method for detecting the presence of a nucleic acid molecule of claim1 in a sample comprising: a) contacting the sample with a nucleic acidprobe or primer which selectively hybridizes to the nucleic acidmolecule; and b) determining whether the nucleic acid probe or primerbinds to a nucleic acid molecule in the sample to thereby detect thepresence of a nucleic acid molecule of claim 1 in the sample.
 13. Themethod of claim 12, wherein the sample comprises mRNA molecules and iscontacted with a nucleic acid probe.
 14. A method for identifying acompound which binds to a polypeptide of claim 6 comprising: a)contacting the polypeptide, or a cell expressing the polypeptide with atest compound; and b) determining whether the polypeptide binds to thetest compound.
 15. The method of claim 14, wherein the binding of thetest compound to the polypeptide is detected by a method selected fromthe group consisting of: a) detection of binding by direct detection oftest compound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor DHDR activity.
 16. A method for identifying a compound whichmodulates the activity of a polypeptide of claim 6 comprising: a)contacting a polypeptide of claim 6 with a test compound; and b)determining the effect of the test compound on the activity of thepolypeptide to thereby identify a compound which modulates the activityof the polypeptide.
 17. The method of claim 16, wherein said activity ismodulation of virus activity.
 18. A method for identifying a compoundwhich modulates virus activity comprising: a) contacting the polypeptideof claim 6, or a cell expressing the polypeptide with a test compound;and b) identifying the compound as a modulator of virus activity bydetermining the effect of the test compound on the activity of thepolypeptide.
 19. The method of claim 16, wherein said activity ismodulation of cellular proliferation.
 20. A method for identifying acompound which modulates cellular proliferation comprising: a)contacting the polypeptide of claim 6, or a cell expressing thepolypeptide with a test compound; and b) identifying the compound as amodulator of cellular proliferation by determining the effect of thetest compound on the activity of the polypeptide.